ADAMTS-5 deficient mice do not develop mechanical allodynia associated with osteoarthritis following medial meniscal destabilization.

OBJECTIVE: To characterize pain-related behavior during the course of knee osteoarthritis (OA) induced by destabilization of the medial meniscus (DMM) in wild type (WT) and in ADAMTS-5 null mice. METHODS: DMM surgery was performed in the right knee of CD-1 mice. At regular intervals up to 8 weeks after surgery, mice were assessed for the following parameters: mechanical allodynia (via withdrawal thresholds to von Frey filaments applied to the plantar surface of both hind paws or to the tail), thermal hyperalgesia, locomotor activity and gait analysis. In addition, mechanical allodynia was tested in C57BL/6 WT or ADAMTS-5 null mice following DMM surgery. RESULTS: In CD-1 mice, a robust and progressive decrease in withdrawal threshold was observed in both hind paws after DMM but not sham surgery. Allodynia was apparent as early as 14 days postoperatively. Both sexes developed OA changes after surgery with concurrent mechanical allodynia. No other pain-related behavioral changes were detected up to 8 weeks post-surgery. In C57BL/6 mice, a genetic background in which only males develop OA changes after DMM, males but not females developed allodynia in the ipsilateral hind paw. In contrast, C57BL/6 ADAMTS-5 null mice did not develop OA changes or mechanical allodynia up to 8 weeks post-surgery. CONCLUSION: Joint pathology following DMM surgery in mice is associated with progressive mechanical allodynia. ADAMTS-5 null mice are resistant to DMM-induced OA-like lesions and to the associated mechanical allodynia.

An immunoaffinity liquid chromatography-tandem mass spectrometry assay for detection of endogenous aggrecan fragments in biological fluids: Use as a biomarker for aggrecanase activity and cartilage degradation.

The degradation of articular cartilage by aggrecanases (ADAMTS-4 and ADAMTS-5) plays a significant role in the pathology of osteoarthritis (OA). To monitor aggrecanase activity in OA, we have developed a sensitive, accurate, and versatile assay for detection of two specific cleavage sites on aggrecan. The assay uses an immunoaffinity-based liquid chromatography-tandem mass spectrometry (LC-MS/MS) method to detect cleavage at the (374)ARGS site and the (1820)AGEG site. The dynamic range of the assay is more than three orders of magnitude, with interassay precision less than 15%. It has been successfully applied to various biological fluids and species, including rat, bovine, dog, and human. The assay has been analytically qualified for use in human urine and synovial fluid (SF). The limits of detection (LODs) for ARGS in urine and SF are 2.5 and 10 pg/ml, respectively, whereas the LOD for AGEG is 20 pg/ml in SF. Analysis of these biomarkers from OA subjects and normal healthy volunteers revealed a significant elevation of both markers in OA. Similarly, in a rat model of cartilage degradation, both ARGS and AGEG were elevated, demonstrating the utility of these biomarkers for translational research. These data suggest that the ARGS and AGEG biomarkers developed have potential as measures of aggrecanase activity in OA and may contribute to our understanding of OA pathology.

Reduced incidence and severity of experimental autoimmune arthritis in mice expressing catalytically inactive A disintegrin and metalloproteinase 8 (ADAM8).

A disintegrin and metalloproteinase 8 (ADAM8), a catalytically active member of the ADAMs family of enzymes, is expressed primarily on immune cells and thus probably involved in inflammatory responses. ADAM8 is also produced by chondrocytes, and recombinant ADAM8 can induce cartilage catabolism. We therefore decided to test the role of ADAM8 in autoimmune inflammatory arthritis using transgenic mice expressing catalytically inactive ADAM8. Transgenic DBA/1J mice expressing an inactivating point mutation in the ADAM8 gene to change Glu330 to Gln330 (ADAM8(EQ)) were generated to evaluate the proteolytic function of ADAM8 in an lipopolysaccharide-synchronized collagen-induced arthritis (LPS-CIA) model of autoimmune arthritis. The systemic inflammatory reaction to LPS was also evaluated in these mice. Expression profiling of paw joints from wild-type mice revealed that ADAM8 mRNA levels increased at the onset of clinical arthritis and correlated well with cellular macrophage markers. When subjected to LPS-CIA, ADAM8(EQ) mice demonstrated decreased incidence and severity of clinical arthritis compared to wild-type mice. Histological examination of paw joints from ADAM8(EQ) mice confirmed marked attenuation of synovial inflammation, cartilage degradation and bone resorption when compared to wild-type mice. However, transgenic mice and wild-type mice responded similarly to LPS-induced systemic inflammation with regard to mortality, organ weights, neutrophil sequestration and serum cytokine/chemokine production. We conclude that ADAM8 proteolytic activity plays a key role in the development of experimental arthritis and may thus be an attractive target for the treatment of arthritic disorders while minimizing risk of immunocompromise.

Purification and cloning of aggrecanase-1: a member of the ADAMTS family of proteins.

We purified, cloned, and expressed aggrecanase, a protease that is thought to be responsible for the degradation of cartilage aggrecan in arthritic diseases. Aggrecanase-1 [a disintegrin and metalloproteinase with thrombospondin motifs-4 (ADAMTS-4)] is a member of the ADAMTS protein family that cleaves aggrecan at the glutamic acid-373-alanine-374 bond. The identification of this protease provides a specific target for the development of therapeutics to prevent cartilage degradation in arthritis.

Aggrecanase. A target for the design of inhibitors of cartilage degradation.

In arthritic diseases there is a gradual erosion of cartilage that leads to a loss of joint function. Aggrecan, which provides cartilage with its properties of compressibility and elasticity, is the first matrix component to undergo measurable loss in arthritis. This loss of aggrecan appears to be due to an increased rate of degradation, that can be attributed to proteolytic cleavage of the core protein within the interglobular domain (IGD). Two major sites of cleavage have been identified within the IGD. One, between the amino acids Asn341-Phe342, where the matrix metalloproteinases (MMPs) have been shown to clip; and the other, between Glu373-Ala374, which is attributed to a novel protease, "aggrecanase." We have generated aggrecanase in conditioned media from IL-1-stimulated bovine nasal cartilage and have used an enzymatic assay to evaluate this proteinase activity. In these studies we follow the generation of aggrecanase and MMPs in response to IL-1 in this system and examine the contribution of these enzymes in aggrecan degredation. Our data suggest that aggrecanase is a key enzyme in cartilage aggrecan degradation that represents a novel target for cartilage protection therapy in arthritis.

Will the real aggrecanase(s) step up: evaluating the criteria that define aggrecanase activity in osteoarthritis.

Loss of aggrecan from articular cartilage is an early and critical event in the pathogenesis of osteoarthritis (OA) and is enzymatically mediated by aggrecanase activity. Since the discovery of aggrecanase-1 (ADAMTS-4) and aggrecanase-2 (ADAMTS-5), both members of the "a disintegrin and metalloproteinase with thrombospondin motif" family of proteinases, other members of the family have been reported to have aggrecanase activity, as currently defined, including ADAMTS-1, -8, -9, -15 and -16. Understanding whether these other ADAMTS members are in fact genuine in vivo aggrecanases will be important for the development of therapeutic agents that aim to block aggrecan degradation. The goal of this review is to look at the current definition of "aggrecanase activity", and define its strengths, weaknesses and suitability for determining which ADAMTS, are aggrecanases that participate in aggrecan catabolism in OA. In addition, we propose a more comprehensive definition of aggrecanase activity, based on 6 criteria that encompass both biochemical and biological characteristics of the endogenous aggrecanase activity detected in vitro and in vivo. Finally, using these criteria, we propose which ADAMTSs should be classified as aggrecanases and therefore be considered as drug targets for the development of chondroprotective OA treatments.

Signal transduction through chondrocyte integrin receptors induces matrix metalloproteinase synthesis and synergizes with interleukin-1.

OBJECTIVE: To study the role of signal transduction via integrin receptors in the production of metalloproteinase by rabbit articular chondrocytes. METHODS: Confluent, primary rabbit articular chondrocytes (RAC) were incubated for 72 hours in the presence of interleukin-1 (IL-1), Arg-Gly-Asp (RGD) peptide, or a combination of IL-1 and RGD peptide. Media were analyzed for stromelysin enzymatic activity using a 3H-labeled transferrin substrate, and for stromelysin and collagenase protein by Western analysis. Gelatinase activity was analyzed by gelatin zymography. IL-1 receptor antagonist (IL-1Ra) protein was used to determine the involvement of IL-1 in mediating the effects of RGD peptide, and fluorescence-activated cell sorter analysis (FACS) was used to examine the effect of IL-1 on chondrocyte integrin subunit expression. RESULTS: RGD peptides induced chondrocyte synthesis of stromelysin, collagenase, and 92-kd gelatinase B, and increased synthesis of the constitutively expressed 72-kd gelatinase A. Further studies focusing on stromelysin demonstrated that this up-regulation was concentration dependent and that RGD peptides synergized with IL-1 in inducing stromelysin synthesis. RGD-induced stromelysin production was inhibited by the IL-1Ra in a concentration-dependent manner, indicating that induction by RGD requires binding of IL-1 to its receptor. FACS analysis of RAC showed that IL-1 stimulation increased the expression of beta 1 and alpha v integrin subunits on the chondrocyte surface. CONCLUSION: Our data demonstrate that signal transduction through chondrocyte integrin receptors up-regulates metalloproteinase expression and that this is likely mediated through induction of IL-1. They also suggest that the binding of adhesion molecules to their chondrocyte integrin receptors reduces the amount of IL-1 required to induce stromelysin synthesis. Up-regulation of chondrocyte integrin expression by IL-1 may play a role in the synergistic effects seen with a combination of IL-1 and RGD peptides. Since elevated levels of both IL-1 and adhesion molecules are present in rheumatoid arthritis and osteoarthritis synovial fluid, our data suggest that this interaction may be important in mediating the cartilage destruction accompanying these diseases.

Age-related changes in aggrecan glycosylation affect cleavage by aggrecanase.

Aggrecan degradation involves proteolytic cleavage of the core protein within the interglobular domain. Because aggrecan is highly glycosylated with chondroitin sulfate (CS) and keratan sulfate (KS), we investigated whether glycosylation affects digestion by aggrecanase at the Glu(373)-Ala(374) bond. Treatment of bovine aggrecan monomers to remove CS and KS resulted in loss of cleavage at this site, suggesting that glycosaminoglycans (GAGs) play a role in cleavage at the Glu(373)-Ala(374) bond. In contrast, MMP-3 cleavage at the Ser(341)-Phe(342) bond was not affected by glycosidase treatment of aggrecan. Removal of KS, but not CS, prevented cleavage at the Glu(373)-Ala(374) bond. Thus, KS residues may be important for recognition of this cleavage site by aggrecanase. KS glycosylation has been observed at sites adjacent to the Glu(373)-Ala(374) bond in steer aggrecan, but not in calf aggrecan (Barry, F. P., Rosenberg, L. C., Gaw, J. U., Gaw, J. U., Koob, T. J., and Neame, P. J. (1995) J. Biol. Chem. 270, 20516-20524). Interestingly, although we found that aggrecanase degraded both calf and steer cartilage aggrecan, the proportion of fragments generated by cleavage at the Glu(373)-Ala(374) bond was higher in steer than in calf, consistent with our observations using aggrecan treated to remove KS. We conclude that the GAG content of aggrecan influences the specificity of aggrecanase for cleavage at the Glu(373)-Ala(374) bond and suggest that age may be a factor in aggrecanase degradation of cartilage.

The role of ADAM-TS4 (aggrecanase-1) and ADAM-TS5 (aggrecanase-2) in a model of cartilage degradation.

INTRODUCTION: Cleavage of aggrecan between residues Glu(373)-Ala(374), which is believed to be a key event in aggrecan destruction in arthritic diseases, has been attributed to an enzymatic activity, aggrecanase. Two cartilage aggrecanases have been identified, aggrecanase-1 (ADAM-TS4) and aggrecanase-2 (ADAM-TS5) and both enzymes have been shown very efficiently to cleave soluble aggrecan at the Glu(373)-Ala(374) site. OBJECTIVE: To determine whether ADAM-TS4 and/or ADAM-TS5 are the aggrecanases responsible for aggrecan catabolism following interleukin-1 (IL-1) and tumor necrosis factor (TNF) treatment of bovine articular cartilage. RESULTS: (1) IL-1- and TNF-stimulated release of aggrecan was associated with cleavage of aggrecan within the C-terminus at the ADAM-TS4 and ADAM-TS5-sensitive sites, Glu(1480)-Gly(1481), Glu(1667)-Gly(1668), and Glu(1871)-Leu(1872). (2) The order of cleavage following IL-1 stimulation of cartilage explants was the same as when soluble aggrecan is digested with recombinant human ADAM-TS4 and ADAM-TS5. (3) Both constitutive and stimulated cleavage of aggrecan at the ADAM-TS4 and ADAM-TS5-sensitive sites in cartilage was blocked by a general metalloproteinase inhibitor but not by a MMP-specific inhibitor, and this inhibition correlated with inhibition of aggrecan release from cartilage. (4) PCR and Western blot analysis indicated that both ADAM-TS proteases are expressed in cartilage explants; ADAM-TS5 is constitutively expressed whereas ADAM-TS4 is induced following IL-1 and TNF treatment. (5) Immunodepletion of both ADAM-TS4 and ADAM-TS5 from bovine articular cartilage cultures following IL-1 stimulation resulted in a 90% reduction of aggrecanase activity in the culture medium.

The interglobular domain of cartilage aggrecan is cleaved by hemorrhagic metalloproteinase HT-d (atrolysin C) at the matrix metalloproteinase and aggrecanase sites.

Two primary cleavage sites have been identified within the interglobular domain of the cartilage aggrecan core protein: one is between amino acid residues Asn 341 and Phe342, where many matrix metalloproteinases (MMP) have been shown to cleave; and the other is between amino acid residues Glu373 and Ala374. Although cleavage at the Glu373-Ala374 site is believed to play a critical role in cartilage aggrecan degradation in arthritic diseases, the enzyme responsible for cleavage at this site, "aggrecanase," has not been identified. Members of the ADAM (a disintegrin and metalloproteinase) family of proteins, which shows structural homology to the snake venom hemorrhagic metalloproteinases (reprolysins), have recently been demonstrated to be expressed in articular chondrocytes. Because many ADAM family members have a putative proteinase function, this raises the possibility that aggrecanase may be a member of this family of proteases. To examine whether reprolysins have the ability to cleave aggrecan at either the aggrecanase site or the MMP site, the snake venom hemorrhagic toxin metalloproteinase HT-d (atrolysin C) was tested for its ability to cleave bovine aggrecan monomer. Cleavage was monitored using the BC-3 antibody, which recognizes aggrecan fragments with the new NH2 terminus ARGSV generated by cleavage at the aggrecanase site, and with the AF-28 antibody, which recognizes aggrecan fragments with the new NH2 terminus FFGVG generated by cleavage at the MMP site. Cleavage at both the aggrecanase and MMP sites occurred in a concentration-dependent manner with 100 nM atrolysin C or greater. AF-28-reactive fragments were generated by 30 min of incubation, and levels were maximal by 8 h; BC-3-reactive fragments were detected at 2 h and continued to increase through 48 h, thus suggesting that atrolysin C can cleave at the MMP and aggrecanase sites. NH2-terminal aggrecan fragments generated by cleavage at the aggrecanase site were also detected using antisera recognizing the new COOH terminus, NITEGE, formed by cleavage at the Glu373-Ala374 bond, indicating that cleavage at this site does not require prior cleavage at the MMP site. These data provide the first demonstration that a reprolysin can cleave the core protein of aggrecan and the first example of a specific protease that can cleave at the aggrecanase site independent of cleavage at the MMP cleavage site.

Cytokine-induced cartilage proteoglycan degradation is mediated by aggrecanase.

OBJECTIVE: To evaluate the relationship between specific cleavage of aggrecan at the Glu373-Ala374 'aggrecanase' site and degradation and release of proteoglycan catabolites from cartilage in explant cultures. DESIGN: The monoclonal antibody, BC-3, which specifically recognizes the new N-terminus, ARGSVIL, generated by cleavage of aggrecan at the Glu373-Ala374 'aggrecanase' site, was used to follow the generation of fragments produced by cleavage at this site as compared to degradation of proteoglycan as assessed by glycosaminoglycan (GAG) release from cartilage in response to cytokines and the ability of inhibitors to block this cleavage. RESULTS: (1) There was a strong correlation between specific cleavage at the Glu373-Ala374 bond and the release of aggrecan catabolites in response to interleukin-1 (IL-1) or tumour necrosis factor (TNF) stimulation. (2) This cleavage in the interglobular domain of aggrecan was inhibited by the inclusion of cycloheximide, thus indicating a requirement for de novo protein synthesis in the induction of 'aggrecanase' activity. (3) The inhibitors, indomethacin, naproxen, tenidap, dexamethasone and doxycycline were ineffective in blocking either specific cleavage at the 'aggrecanase' site or aggrecan degradation as measured by GAG release from cartilage. (4) In contrast, compounds which act through two different mechanisms to inhibit MMPs were effective in blocking both specific cleavage at the 'aggrecanase' site and proteoglycan degradation. CONCLUSIONS: Our data suggest that 'aggrecanase' is primarily responsible for proteoglycan cleavage in these experimental systems and that this protease has properties in common with metalloproteases including members of the MMP and ADAM family. Inhibition of 'aggrecanase' may have utility in preventing cartilage loss in arthritis.

Cleavage of native cartilage aggrecan by neutrophil collagenase (MMP-8) is distinct from endogenous cleavage by aggrecanase.

Cleavage of aggrecan core protein at the Glu373-Ala374 site by the unidentified enzyme, "aggrecanase," is thought to play an important role in cartilage degradation. To examine aggrecan cleavage by MMP-8 at this aggrecanase site, we evaluated the release of fragments with the N terminus ARGSVIL from freeze-thawed bovine nasal cartilage using the monoclonal antibody BC-3. Recombinant human MMP-8 catalytic domain cleaved native aggrecan in a concentration-related manner between 0.2 and 2 microg/ml, with complete release of glycosaminoglycan at 2 microg/ml or greater. Cleavage at the aggrecanase site was observed only at MMP-8 concentrations resulting in complete release of glycosaminoglycan from the cartilage, suggesting that preferential cleavage occurs at a different site. Time course studies indicated that only following depletion of substrate containing the preferred clip site did MMP-8 rapidly cleave at the aggrecanase site. Finally, MMP-8 resulted in a different pattern of BC-3-reactive fragments from that produced by endogenous aggrecanase in live cartilage, and SA751(N-(1(R)-carboxyethyl) -alpha-(S)-(4-phenyl-3-butynyl)glycyl-L-O-methyltyrosine, N-methylamide), a potent inhibitor of MMP-8 (Ki = 2 nM) which was effective in blocking cleavage by MMP-8 at the aggrecanase site with an IC50 in the nanomolar range, did not prevent aggrecan degradation or specific cleavage at this site by endogenously generated aggrecanase at concentrations up to 100 microM. Taken together these data suggest that MMP-8 does not represent cartilage aggrecanase.

Generation and characterization of aggrecanase. A soluble, cartilage-derived aggrecan-degrading activity.

A method was developed for generating soluble, active "aggrecanase" in conditioned media from interleukin-1-stimulated bovine nasal cartilage cultures. Using bovine nasal cartilage conditioned media as a source of the aggrecanase enzyme, an enzymatic assay was established employing purified aggrecan monomers as a substrate and monitoring specific aggrecanase-mediated cleavage products by Western analysis using the monoclonal antibody, BC-3 (which recognizes the new N terminus, ARGS, on fragments produced by cleavage between amino acid residues Glu373 and Ala374). Using this assay we have characterized cartilage aggrecanase with respect to assay kinetics, pH and salt optima, heat sensitivity, and stability upon storage. Aggrecanase activity was inhibited by the metalloprotease inhibitor, EDTA, while a panel of inhibitors of serine, cysteine, and aspartic proteinases had no effect, suggesting that aggrecanase is a metalloproteinase. Sensitivity to known matrix metalloproteinase inhibitors as well as to the endogenous tissue inhibitor of metalloproteinases, TIMP-1, further support the notion that aggrecanase is a metalloproteinase potentially related to the ADAM family or MMP family of proteases previously implicated in the catabolism of the extracellular matrix.

Sites of aggrecan cleavage by recombinant human aggrecanase-1 (ADAMTS-4).

Aggrecan, the major proteoglycan of cartilage that provides its mechanical properties of compressibility and elasticity, is one of the first matrix components to undergo measurable loss in arthritic diseases. Two major sites of proteolytic cleavage have been identified within the interglobular domain (IGD) of the aggrecan core protein, one between amino acids Asn(341)-Phe(342) which is cleaved by matrix metalloproteinases and the other between Glu(373)-Ala(374) that is attributed to aggrecanase. Although several potential aggrecanase-sensitive sites had been identified within the COOH terminus of aggrecan, demonstration that aggrecanase cleaved at these sites awaited isolation and purification of this protease. We have recently cloned human aggrecanase-1 (ADAMTS-4) (Tortorella, M. D., Burn, T. C., Pratta, M. A., Abbaszade, I., Hollis, J. M., Liu, R., Rosenfeld, S. A., Copeland, R. A., Decicco, C. P., Wynn, R., Rockwell, A., Yang, F., Duke, J. L., Solomon, K., George, H., Bruckner, R., Nagase, H., Itoh, Y., Ellis, D. M., Ross, H., Wiswall, B. H., Murphy, K., Hillman, M. C., Jr., Hollis, G. F., Newton, R. C., Magolda, R. L., Trzaskos, J. M., and Arner, E. C. (1999) Science 284, 1664-1666) and herein demonstrate that in addition to cleavage at the Glu(373)-Ala(374) bond, this protease cleaves at four sites within the chondroitin-sulfate rich region of the aggrecan core protein, between G2 and G3 globular domains. Importantly, we show that this cleavage occurs more efficiently than cleavage within the IGD at the Glu(373)-Ala(374) bond. Cleavage occurred preferentially at the KEEE(1667-1668)GLGS bond to produce both a 140-kDa COOH-terminal fragment and a 375-kDa fragment that retains an intact G1. Cleavage also occurred at the GELE(1480-1481)GRGT bond to produce a 55-kDa COOH-terminal fragment and a G1-containing fragment of 320 kDa. Cleavage of this 320-kDa fragment within the IGD at the Glu(373)-Ala(374) bond then occurred to release the 250-kDa BC-3-reactive fragment from the G1 domain. The 140-kDa GLGS-reactive fragment resulting from the preferential cleavage was further processed at two additional cleavage sites, at TAQE(1771)-(1772)AGEG and at VSQE(1871-1872)LGQR resulting in the formation of a 98-kDa fragment with an intact G3 domain and two small fragments of approximately 20 kDa. These data elucidate the sites and efficiency of cleavage during aggrecan degradation by aggrecanase and suggest potential tools for monitoring aggrecan cleavage in arthritis.

Cloning and characterization of ADAMTS11, an aggrecanase from the ADAMTS family.

Aggrecan is responsible for the mechanical properties of cartilage. One of the earliest changes observed in arthritis is the depletion of cartilage aggrecan due to increased proteolytic cleavage within the interglobular domain. Two major sites of cleavage have been identified in this region at Asn(341)-Phe(342) and Glu(373)-Ala(374). While several matrix metalloproteinases have been shown to cleave at Asn(341)-Phe(342), an as yet unidentified protein termed "aggrecanase" is responsible for cleavage at Glu(373)-Ala(374) and is hypothesized to play a pivotal role in cartilage damage. We have identified and cloned a novel disintegrin metalloproteinase with thrombospondin motifs that possesses aggrecanase activity, ADAMTS11 (aggrecanase-2), which has extensive homology to ADAMTS4 (aggrecanase-1) and the inflammation-associated gene ADAMTS1. ADAMTS11 possesses a number of conserved domains that have been shown to play a role in integrin binding, cell-cell interactions, and extracellular matrix binding. We have expressed recombinant human ADAMTS11 in insect cells and shown that it cleaves aggrecan at the Glu(373)-Ala(374) site, with the cleavage pattern and inhibitor profile being indistinguishable from that observed with native aggrecanase. A comparison of the structure and expression patterns of ADAMTS11, ADAMTS4, and ADAMTS1 is also described. Our findings will facilitate the study of the mechanisms of cartilage degradation and provide targets to search for effective inhibitors of cartilage depletion in arthritic disease.