And ADAM-TS5 (Aggrecanase-2) in a Model of Cartilage Degradation M

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Osteoarthritis and Cartilage (2001) 9, 539–552 © 2001 OsteoArthritis Research Society International 1063–4584/01/060539+14 $35.00/0 doi:10.1053/joca.2001.0427, available online at http://www.idealibrary.com on

The role of ADAM-TS4 (aggrecanase-1) and ADAM-TS5 (aggrecanase-2) in a model of cartilage degradation M. D. Tortorella*, A.-M. Malfait*, C. Deccico and E. Arner† DuPont Pharmaceuticals Company, Wilmington, DE 19880-0400, USA

Summary

Introduction: Cleavage of aggrecan between residues Glu373–Ala374, 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 Glu373–Ala374 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, Glu1480–Gly1481, Glu1667–Gly1668, and Glu1871–Leu1872. (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. © 2001 OsteoArthritis Research Society International Key words: Aggrecan, Cartilage, Aggrecanase, ADAM-TS.

Introduction the loss of aggrecan from the matrix is a key event in cartilage destruction. Indeed, one of the earliest changes Cartilage degradation is the hallmark of rheumatoid arthritis observed in osteoarthritis is the loss of aggrecan from the (RA) and osteoarthritis (OA). The major components of the cartilage matrix3. cartilage extracellular matrix are type II collagen and aggre- Aggrecan depletion in arthritic cartilage has been can. Aggrecan monomers are approximately 2.5 million ascribed to increased proteolytic cleavage of the core Daltons and consist of a 250-kD protein core with chondroi- protein. Two major cleavage sites have been identiﬁed in tin sulfate and keratan sulfate glycosaminoglycan (GAG) the IGD, one at Asn341–Phe342 and one at the Glu373– side chains attached to it. The amino terminal region of the Ala374 bond. Matrix metalloproteinases (MMP) act primarily aggrecan core protein contains two globular domains, G1 on the former bond and all MMPs found to be present in and G2, which are separated by an interglobular domain cartilage including MMP-1, MMP-2, MMP-3, MMP-7, (IGD) that spans about 150 amino residues. The G2 region MMP-8, MMP-9, MMP-13, and MT1-MMP, are capable of is followed by a long GAG attachment region and by the cleaving aggrecan at this site in vitro4–7. Cleavage at 1,2 carboxyl terminal globular domain G3 . Aggrecan mono- the second site, Glu373-Ala374, has been attributed to an mers interact with hyaluronan through their G1 domain and enzymatic activity referred to as aggrecanase8–11. thus form large aggregates containing 10–100 aggrecan Since the discovery of aggrecanase activity, we have monomers on a hyaluronan backbone. Because aggrecan identiﬁed and cloned two cartilage aggrecanases, provides cartilage with its properties of compressibility and aggrecanase-112 and aggrecanase-213. Both enzymes are elasticity by swelling and hydrating the collagen framework, members of the a disintegrin and metalloprotease (ADAM) family of zinc metalloproteases that consist of an amino- Received 18 October 2000; revision requested 12 December terminal propeptide domain, a metalloproteinase domain, 2000; revision received 16 January 2001; accepted 17 January and a disintegrin-like domain, resembling the structural 2001. elements of the reprolysin family of metalloproteases14. *Both authors contributed equally to study. Unlike typical ADAM proteins that are membrane-anchored This work was supported by the Wellcome Trust Grant and have a transmembrane and a cytoplasmic domain in No 057508 the carboxyl terminal region, the C-terminal half of Address correspondence to: Micky Tortorella, Pharmacia Corpor- aggrecanase-1 and aggrecanase-2 contains a varying ation, Skokie Discovery Biology, 4901 Searle Parkway, Room J-245, Skokie, IL 60077, USA. Tel: (1) 847 982 7517; Fax: (1) 847 number of thrombospondin (TS) type 1 motifs, the 982 7680; E-mail: [email protected] sequence of which is the conserved motif in thrombo- †Present address: Pharmacia Corporation, Skokie Discovery spondin 1 and 2. ADAM proteins containing these TS motifs Biology, 4901 Searle Parkway, Room J-200A, Skokie, IL 60077. are referred to as ADAM-TS proteins, and aggrecanase-1

539 540 M. D. Tortorella et al.: ADAM-TS4/TS5 in cartilage aggrecan breakdown a

Fig. 1. Schematic representation of MMP- and ADAM-TS4- and ADAM-TS5-generated fragments of bovine aggrecan. (a) ADAM-TS4 and ADAM-TS5 cleavage sites within bovine aggrecan. (b) MMP- and ADAM-TS-generated fragments. and aggrecanase-2 have been designated ADAM-TS4 and fragments previously reported to be released during ADAM-TS5, respectively. IL-1alpha-treatment of bovine articular cartilage suggesting Both enzymes have been shown to cleave within the IGD cleavage at the Glu1771–Ala1772 and Glu1871–Leu1872 at the aggrecanase site between residues Glu373 and bonds16,17. Cleavage of cartilage aggrecan within the IGD Ala374, but not at the MMP site between residues Asn341 between amino acids Glu373 and Ala374 results in the and Phe342. In addition, we have recently demonstrated formation of a small N-terminal fragment with a new that human recombinant ADAM-TS4 and ADAM-TS5 both C-terminus NITEGE373 and a large C-terminal fragment cleave aggrecan preferentially at four sites located in the with the new N-terminus 374ARGSV. Researchers have chondroitin sulfate-rich region between G2 and G3 at the designed antibodies to these neoepitopes and are currently Glu1480–Gly1481, Glu1667–Gly1668, Glu1771–Ala1772 and using them to monitor aggrecanase-mediated cleavage. Glu1871–Leu1872 bonds (15 and Tortorella et al., in prep- However, using these antibodies to confirm that ADAM- aration) (Fig. 1). Two of these sites correspond with TS4/TS5 are responsible for endogenous aggrecanase Osteoarthritis and Cartilage Vol. 9, No. 6 541 activity is complicated by the potential for cleavage at the Gly1481 bond, and at the Glu1667–Gly1668 bond, Glu373–Ala374 bond by MMP-818 and MT1-MMP19. There- respectively, as described15. A polyclonal neoepitope anti- fore, in the present study, we investigated whether MMP-8 body was also prepared to the peptide sequence and MT1-MMP cleave aggrecan at the ADAM-TS4/TS5 1872LGQRPPVT that is present on the N-terminus of aggre- preferential sites of cleavage within the C-terminus of can fragments generated by cleavage at the Glu1871– aggrecan. Furthermore, we employed neoepitope anti- Leu1872 bond [Fig. 1(a)]. Specificity of the antibodies was bodies to the products of ADAM-TS4/5 cleavage in the determined and intact undigested aggrecan was not C-terminus of aggrecan, selective inhibitors, evaluation of detected with any of the antibodies15. In bovine aggrecan, ADAM-TS4/TS5 message and protein, and immuno- cleavage at the Glu1480-Gly1481 bond generates the depletion to elucidate whether these aggrecanases are C-terminal neoepitope GELE rather than the SELE critical for the breakdown of aggrecan during IL-1- and neoepitope of the human aggrecan fragment with a single TNF-induced degradation of articular cartilage explants. amino acid substitution of G for S in the P4 position. In addition, the IPEN C-terminal neoepitope generated by cleavage at the Asn341–Phe342 bond in the IGD of human Method aggrecan is IPES in bovine aggrecan with a single amino acid substitution of N for S at the P1 position. The antibody MATERIALS raised against the GELE neoepitope does not recognize the C-terminus SELE and the antibody raised against the Dulbecco’s modified Eagle’s medium (DMEM), fetal calf IPES neoepitope does not recognize the C-terminus IPEN serum (FCS) and penicillin/streptomycin were purchased on the human aggrecan fragment illustrating the high from BioWhittakers (Verviers, Belgium). IL-1 and IL-1 degree of specificity of these antibodies. receptor antagonist (IL-1ra) were a gift from Dr Jerry Saklatvala (Kennedy Institute of Rheumatology, London, U.K.)20. TNF was a gift from Professor W. Stec (Centre of Molecular and Macromolecular Biology, Lodz, Poland) ADAM-TS ANTIBODIES and neutralizing anti-TNF antibodies (A2) were a gift Polyclonal antibodies for ADAM-TS5, ADAM-TS4 and from Centocor Inc (Malvern, PA, U.S.A.). Keratanase, ADAM-TS1 were prepared to the peptide sequences keratanase II and chondroitinase ABC were from ILTSIDASKP (residues 502–511), VMAHVDPEEP (resi- Seikagaku (Kogyo, Japan). Full length MMP-8 were cloned dues 502–511) and DAKQCASLNG (residues 481–490), and expressed in E. coli at DuPont Pharmaceuticals. MT1- respectively12,13. The antibodies were all purified over a MMP was a gift from Dr Duanqing Pei (University of protein G-Sepharose column and the protein concentration Minnesota, Minneapolis, MN). Antibody BC-321 that was determined using BCA reagent (Pierce, Rockford, IL). recognizes the new N-terminus ARGS on aggrecan frag- The immunizing peptide for each antibody blocked bind- ments produced by cleavage at the Glu373–Ala374 bond, ing to the antigen and the antibody to ADAM-TS1 did was licensed from Dr Bruce Caterson (University of Wales, not cross-react with ADAM-TS4/TS5, the antibody to Cardiff, U.K.). Antibody AF-2822 that recognizes the new ADAM-TS4 did not cross-react with ADAM-TS5, and N-terminus, FFGVG, on aggrecan fragments produced by finally, the antibody to ADAM-TS5 did not cross-react with cleavage at the Asn341–Phe342 bond, was a gift from ADAM-TS4 (Fig. 2). Dr Amanda Fosang (University of Melbourne, Parkville, Australia). The hydroxamic acids XS309 ([3S-[3R*, 2-[2R*,2-(R*,S*)]-hexahydro-2-(2-[2-(hydro xyamino)-1- methyl-2-oxyethyl]-4-methyl-1-oxypentyl]-N-methyl-3-p RT-PCR FOR ADAM-TS4 AND ADAM-TS5 yridazinecarboxamide) and SE206 (2S,2R,6S-3-aza-4- oxo-10-oxa-5-hexyl-2-(methylcarboxamido)-10-pa racyclo- RNA was isolated from cartilage using RNeasy phane-6-N hydroxycarboxamide) were synthesized at Mini-columns (QIAGEN Ltd, U.K.) and then converted into DNA using Superscript II (Gibco). RT-PCR reactions were DuPont Pharmaceuticals23,24. The IC values of these 50 performed using Ready-To-Go PCR beads (Amersham compounds against MMP-1, -2, -3, -8, -9 and 14 were Pharmacia, Piscataway, NJ). For ADAM-TS4 the primers calculated using the fluorogenic peptide substrate, Mca- used were 5′-ACCACTTTGACACAGCCATTC-3′ (forward, Pro-Leu-Gly-Leu-Dpa-Ala-Arg-NH , at a concentration of 2 catalytic domain) and 5′-ACCCCCACAGGTCCGAGA 1 M, and those compounds against ADAM-TS4 and GCA-3′ (reverse, thrombospondin motif). For ADAM-TS5 ADAM-TS5 were determined using 500 mM purified bovine the primers used were 5′-TGTGCTGTGATTGAAGAC nasal aggrecan as previously described25. XS309 is a GAT-3′ (forward, catalytic domain) and 5′-GACTGCAGGA nanomolar inhibitor of a large number of MMPs, but is GCGGTAGATGG-3′ (reverse, thrombospondin motif). For inactive at >10 M in blocking ADAM-TS4/TS5. In con- bovine glyceraldehyde phospate dehydrogenase (GAPDH) trast, SE206 is a nanomolar inhibitor of both MMPs and the primers used were 5′-TGGTATCGTGGAAGGACT ADAM-TS4/TS5. The IC value of SE206 against 50 CAT-3′ (forward) and 5′-GTGGGTGTCGCTGTTGAA ADAM-TS4 is 76 nM12, and against ADAM-TS513 is 40 nM. GTC-3′ (reverse). The primers for ADAM-TS4 and ADAM-TS5, ADAM-TS4 and ADAM-TS1 were cloned and ADAM-TS5 are derived from the human sequence, and expressed in Drosophila S2 cells as described12,13. were found to cross react with bovine cDNA.

NEOEPITOPE ANTIBODIES BOVINE ARTICULAR CARTILAGE CULTURE Polyclonal neoepitope antibodies were prepared to the peptide sequences DFVDIPES, PRNITEGE, ATTAGELE Articular cartilage was dissected from the knees of and PTTFKEEE that are present on the C-terminus of 6-year-old cows. Cartilage was allowed to equilibrate for aggrecan fragments generated by cleavage at the Ser341– 3 days in DMEM supplemented with 10% FCS, penicillin Phe342 bond, at the Glu373–Ala374 bond, at the Glu1480– (100 U/ml) and streptomycin (100 g/ml). Subsequently, 542 M. D. Tortorella et al.: ADAM-TS4/TS5 in cartilage aggrecan breakdown

Fig. 2. Monospeciﬁcity of antisera to ADAM-TS4 and ADAM-TS5. Twenty ng of ADAM-TS4 (panel A) or ADAM-TS5 (panel B) were separated by SDS-PAGE, transferred to PVDF membranes, and incubated with antibodies at a concentration of 10 g/ml to (A) ADAM-TS1, (B) ADAM-TS4, (C) ADAM-TS5 (D) ADAM-TS4 with 100 M of peptide VMAHVDPEEP, (E) ADAM-TS4 with 100 M of peptide ILTSIDASKP, (F) ADAM-TS5 with 100 M of peptide VMAHVDPEEP, and (G) ADAM-TS5 with 100 M of peptide ILTSIDASKP. cartilage was cut into 3×3 mm explants, weighing approxi- umes of acetone and reconstituted in 30 l of Tris glycine/ mately 10–20 mg each, and incubated in 96-well plates for SDS sample buffer containing 2.5% -mercaptoethanol 72 h with either control medium (serum-free DMEM), IL-1 and heated for 3 min at 100°C. (100 ng/ml), TNF (100 ng/ml), IL-1 (100 ng/ml) plus IL-1ra (10 g/ml), or TNF (100 ng/ml) plus anti-TNF antibodies (10 g/ml). For the inhibition studies with SE206 and WESTERN BLOT ANALYSIS XS309, cartilage was dissected from the knees of calves and incubated in 96-well plates for 72 h with either control Culture media and cartilage extracts were analysed by medium, IL-1 (100 ng/ml), IL-1 (100 ng/ml) plus XS309 SDS-PAGE on 4–12% polyacrylamide gels under reducing (10 M), or IL-1 (100 ng/ml) plus SE206 (10 M). conditions. When analysing culture media for aggrecan catabolites, equal volumes (20 l, i.e. 10% of the culture medium) were loaded. When analysing cartilage matrix EXTRACTION OF AGGRECAN AND ITS CATABOLITES FROM extracts for aggrecan catabolites, gels were loaded on an CARTILAGE equal GAG basis (20 g). The separated proteins were transferred to PVDF membranes and immunolocalized with At the end of the 72 h culture period, proteoglycans were a 1:1000 dilution of one of the neoepitope antibodies or extracted from the cartilage explants for 48 h at 4°C with with the antibodies against ADAM-TS4 or ADAM-TS5. 4 M guanidinium hydrochloride, 50 mM sodium acetate Subsequently, the membranes were incubated with a buffer, pH 6.8, containing disodium EDTA (0.01 M), 1:5000 dilution of goat antimouse or goat antirabbit IgG 6-aminohexanoic acid (0.1 M), benzamidine hydrochloride alkaline phosphatase conjugate as the secondary antibody. (0.05 M), phenylmethanesulfonylﬂuoride (2 mM). Extracts Products were visualized by developing the blots in were then dialysed in water. 5-bromo-4-chloro-3-indolyl-1-phosphate nitro blue tetrazo- lium color developing reagent. Overnight transfer resulted in complete transfer of both low and high molecular GLYCOSAMINOGLYCAN ASSAY fragments. For monitoring the order in which 374ARGS-, 1480 1667 Glycosaminoglycan (GAG) levels in the culture media GELE - and KEEE -reactive fragments are gener- and cartilage extracts were determined by the dimethyl- ated in cartilage following IL-1 stimulation, conditions of methylene blue (DMMB) assay, as described by Farndale the Western blot analysis were adjusted to provide equal et al.26. Shark chondroitin sulfate was used as a standard, intensity of staining for equivalent amounts of antigen. The antibodies exhibited different affinities for their respective and results were expressed as g of GAG per mg of wet weight cartilage. peptide antigens. Therefore, in order to attain equal reactivity for the Western analysis, the dilution of each antibody that yielded an optical density (OD) of 0.5 was DEGLYCOSYLATION OF AGGRECAN AND AGGRECAN CATABOLITES used. This antibody dilution was determined by ELISA, as follows. The immunizing peptide for each antibody was Proteoglycans and their breakdown products were linked to biotin at the N-terminus or C-terminus and was digested with keratanase (0.01 units per g GAG), then immobilized at a concentration of 10 nM on a 96-well keratanase II (0.001 units per g GAG), and chondroitinase streptavidin-coated plate. Varying concentrations of each ABC (0.01 units per g GAG) in buffer containing 50 mM antibody, ranging from 1:10 to 1:100,000 dilutions, were sodium acetate, 0.1 M Tris-HCl, pH 6.5, at 37°C for 3 h. added, followed by a rabbit IgG second antibody conju- After digestion the aggrecan was precipitated with 5 vol- gated to alkaline phosphatase. Binding was assessed Osteoarthritis and Cartilage Vol. 9, No. 6 543

Table I Cleavage of aggrecan by metalloproteinases*† Protease MMP-epitopes Aggrecanase-derived epitopes IPES FFGV TEGE ARGS KEEE GLGS GELE AGEG LGQR ADAM-TS4 —— +++++++ ADAM-TS5 —— +++++++ MMP-8 + + + + ———— — MT1-MMP + + ———————

*Metalloproteinases (100 nM) and ADAM-TS proteins (10 nM) were incubated with 500 nM of aggrecan for 24 h at 37°C. Following the incubation, the products were analysed for MMP-generated neoepitopes and aggrecanase-generated neoepitopes by Western blot analysis. †+Indicates presence and — indicates absence of neoepitope-reactive bands. using 4-nitrophenyl phosphate, disodium salt hexahydrate; for products formed by cleavage at the Glu373–Ala374 bond para-nitrophenyl phosphate substrate (pNPP) and monitor- using a BC-3 ELISA. The efficiency of immunoprecipitation ing OD at 405 nm. The dilution that yielded an OD of 0.5 for has been determined for each antibody. Both the ADAM- the KEEE antibody was a 1:20,000 dilution, for the GELE TS4 and ADAM-TS5 antisera successfully removed 100 ng antibody a 1:10,000 and for the BC-3 antibody a 1:1000 of enzyme that was added to 1 ml of DMEM medium. In dilution. Biotin was incorporated to the N terminus of the order to determine the efficiency of the ADAM-TS1 immuno- ATTAGELE and PTTFKEEE peptides using the solid phase precipitation, we looked at removal of enzyme by Western step-wise synthesis (SPSS) process on an automated Blot analysis, and we achieved removal of >95% of synthesizer. The completed peptide chain (synthesized by ADAM-TS1 from DMEM medium. standard Fmoc chemistry) was first extended at the N terminus with one residue of a 7-atom spacer molecule (6-aminohexanoic acid). This was followed by addition of Results the biotin to the spacer. The reaction is a condensation between the carboxyl of the biotin and the N-amino of the CLEAVAGE OF AGGRECAN BY METALLOPROTEINASES 6-aminohexanoic acid using standard activation chemistry. First, we wanted to determine whether MMP-8 or The biotin was used in the same mass excess as the amino MT1-MMP could cleave cartilage aggrecan at the acids for the synthesis of the peptide. As biotin has low ADAM-TS4/TS5-preferred sites, between Glu373 and solubility in peptide synthesis solvents it was pre-dissolved Ala374, Glu1480 and Gly1481, Glu1667 and Gly1668, Glu1771 in DMSO before delivery to the reaction vessel. The com- and Ala1772, and Glu1871 and Leu1872. In these studies plete biotinylated peptide was cleaved from the solid phase MMP-8 and MT1-MMP were incubated at a concentration and fully deprotected using standard methods. The product of 100 nM with 500 nM of bovine nasal aggrecan for 2 and was precipitated with methyl tertiary butyl ether (MTBE), 24hat37°C. As expected, they cleaved aggrecan between washed twice with MTBE, dried, dissolved in water and residues Ser341 and Phe342 to generate DIPES341 and analysed by RPHPLC (C8). Purification was performed 342FFGVG neoepitopes, which were detected at both 2 and by RPHPLC (C8) using water/acetonitrile gradient with 24 h. However, neither of the MMPs under these exper- trifluoracetic acid as counter ion. Collected fractions were imental conditions were able to produce any of the analysed by RPHPLC (C8) and an homogeneous pool was aggrecanase-derived neoepitopes with the exception of formed. This was freeze-dried and sealed under vacuum. MMP-8, which cleaved aggrecan in the interglobular The mass of the product was confirmed by MALDIMS. domain between residues Glu373 and Ala374 to generate a C-terminal biotinylation of the ARGSVIL peptide was done NITEGE373 and 374ARGS aggrecan fragment at 24 h at QCB (Hopkinton, MA). (Table I). However, MMP-8 did not cleave the C-terminal bonds that are readily hydrolysed by ADAM-TS4 and ADAM-TS5. Therefore, neoepitope antibodies to aggrecan IMMUNODEPLETION OF ADAM-TS1, ADAM-TS4, AND ADAM-TS5 fragments generated by cleavage at the ADAM-TS4/TS5 1.5 g of rabbit polyclonal antibody for ADAMTS-1, preferred sites provide a more specific tool for studying ADAMTS-4 or ADAMTS-5 was coupled to 25 l of protein the involvement of aggrecanases in cartilage aggrecan G-Sepharose (Amersham-Pharmacia). 200 l of culture breakdown. media from the 72 h treated bovine articular cartilage explants, containing endogenous aggrecanase activity were incubated with the antibody-coupled Sepharose for IL-1- OR TNF-INDUCED GAG RELEASE FROM CARTILAGE EXPLANTS 2 h at 4°C with constant rotation in a tube rotator. The CORRELATES WITH AGGRECANASE-GENERATED BUT NOT samples were then centrifuged and the immuno-depleted MMP-GENERATED NEOEPITOPES FROM AGGRECAN CLEAVAGE media were removed. In order to remove pre-existing WITHIN THE INTERGLOBULAR DOMAIN aggrecan fragments from the media the material was next passed over a Q5-anion exchange membrane adsorber Cartilage explants were cultured in the absence or (Sartorius, Goettingen, Germany). Both ADAM-TS4 and presence of IL-1, TNF, IL- plus IL-1ra, or TNF plus ADAM-TS5 have been shown not to bind to a Q-column, neutralizing anti-TNF antibody. After 72 h, media were with a recovery of 92% and 90%, respectively. The collected for the measurement of GAG content. As ADAM-TS-depleted medium were then assessed for expected, both IL-1 and TNF induced an increased aggrecanase activity by incubating with purified aggrecan release of GAG into the culture medium, as a result of (500 nM) for 4 h at 37°C in 50 mM Tris, 150 mM NaCl, degradation of aggrecan (Table II). IL-1-induced GAG 10 mM CaCl, pH 7.5 buffer and the digests were monitored release was inhibited by the addition of excess IL-1ra, and 544 M. D. Tortorella et al.: ADAM-TS4/TS5 in cartilage aggrecan breakdown

Table II (B-fragment) was present in the culture media of both Cytokine-induced degradation of aggrecan in bovine articular IL-1- and TNF-stimulated cartilage [Fig. 3(b)]. cartilage explants* The cartilage extracts were then analysed for the G1 341 Treatment GAG Levels fragments with the MMP-generated IPES neoepitope 373 (g/mg) and aggrecanase-generated TEGE neoepitope. Basal levels of the IPES341 neoepitope were detected in unstimu- Control 1.8 lated cartilage, seen as a triplet near ∼64 kDa (C-fragment) IL-1 [100 ng/ml] 19.5 and no significant induction of this epitope occurred with a TNF [100 ng/ml] 18.8 72-hour cytokine stimulation (Fig. 3(c)]. To eliminate the IL-1+IL-1ra [10 g/ml] 4.7 TNF+anti-TNF [10 g/ml] 2.9 possibility that FFGV-containing fragments accumulate in the cartilage extracellular matrix, we analysed the extracts for these fragments (A-fragment) and could not detect any *Bovine cartilage explants were incubated for 72 h with 373 cytokines at the specified concentrations. At the end of the culture (data not shown). The TEGE neoepitope, seen as a period, media were analysed for GAG content by DMMB assay. doublet between 90 kDa and 64 kDa (D-fragment), was not present in unstimulated cartilage, but was strongly induced by IL-1 and to a lesser degree by TNF [Fig. 3(d)]. The actions of both IL-1 and TNF in inducing cleavage at the the action of TNF by the neutralizing anti-TNF antibody, 373 374 thus demonstrating that the observed cytokine effects were aggrecanase-sensitive Glu –Ala bond within the IGD specific. were abolished by the addition of IL-1ra and anti-TNF- These same culture media were analysed for cleavage antibody, respectively. These results confirm that the GAG of aggrecan within the IGD at the aggrecanase site release observed after a 72-h stimulation with IL-1 or TNF between residues Glu373 and Ala374 and at the MMP site, is mediated by the cleavage of aggrecan within the IGD by between residues Asn341 and Phe342. Using the mono- aggrecanase and not MMP activity. clonal antibodies BC-3 and AF-28, the culture media were analysed for aggrecan products with the MMP-generated ADAM-TS4/TS5-GENERATED NEOEPITOPES WITHIN THE 342 FFGV N-terminus and aggrecanase-generated C-TERMINUS OF AGGRECAN 374ARGS N-terminus. No FFGV-reactive fragments (A-fragment) were detected [Fig. 3(a)], but an array of Next, we evaluated release of specific neoepitopes aggrecan fragments with the N-terminus ARGS generated by ADAM-TS4/TS5 cleavage within the region ab

Fig. 3. Cytokine-induced degradation of aggrecan in bovine articular cartilage explants. Bovine cartilage explants were incubated for 72 h with (a) control media or media containing either (b) IL-1 (100 ng/ml), (c) TNF (100 ng/ml), (d) IL-1 plus IL-1ra (10 g/ml), or (e) TNF plus neutralizing antibodies (10 g/ml). At the end of the culture period, media were analysed for aggrecan fragments containing (a) the N-terminus 342FFGV or (b) 374ARGS, by Western blot analysis on 4–12% gels loaded on an equal volume basis. Next, aggrecan and its catabolites were extracted from the matrix and analysed for aggrecan fragments containing (c) the DIPES341 epitope and (d) the NITEGE373 epitope by Western blot analysis on 4–12% gels loaded on an equal GAG basis. Osteoarthritis and Cartilage Vol. 9, No. 6 545 ab

Fig. 4. Cytokine-induced degradation of aggrecan in bovine articular cartilage explants. Bovine cartilage explants were incubated for 72 h with (a) control media or in media containing either (b) IL-1 (100 ng/ml), (c) TNF (100 ng/ml), (d) IL-1 plus IL-1ra (10 g/ml), or (e) TNF plus neutralizing antibodies (10 g/ml). At the end of the culture period, media were analysed for aggrecan fragments (a) containing the N-terminus KEEE1667 by Western blot analysis on 4–12% gels loaded on an equal volume basis. The higher molecular weight KEEE-reactive band corresponds to the E-fragment, and the lower molecular weight fragment corresponds to the F-fragment. Western Blot for the GELE1480 epitope was performed on both the (b) matrix, on an equal GAG basis, and the (c) media, on an equal volume basis. Finally, (d) fragments containing the N-terminus 1872LGQR were detected in the culture media. between the G2 and G3 domain, following IL-1 and 250 kDa fragment in the culture media following IL-1 and TNF stimulation. The most preferred cleavage site of TNF stimulation [Fig. 4(c)]. The 350 kDa band most prob- ADAM-TS4 and ADAM-TS5 is between Glu1667 and ably represents a fragment with an intact G1 domain Gly1668 (15 and Tortorella et al., in preparation). To evaluate (G-fragment), consistent with it being retained in the matrix cleavage at this site, the culture media were analysed for bound to hyaluronan, and the 250 kDa band is a fragment KEEE1667 containing aggrecan products by Western analy- with the 374ARGS amino terminus (H-fragment). The sis. We detected a 375 kDa fragment that appeared as a formation of these fragments was also blocked by IL-1ra doublet in the IL-1 and TNF-stimulated cultures [Fig. 4(a)]. and by the TNF neutralizing antibody. Interestingly, unlike The higher molecular weight band most probably repre- the ARGS-Western Blot [Fig. 3(b)], the GELE blots reveal sents a KEEE fragment with an intact G1 domain (E- just two speciﬁc fragments, G and H, and no smaller fragment), while the lower band corresponds to a fragment molecular weight fragments containing the GELE epitope with the 374ARGS amino terminus (F-fragment). However, are detected. These data suggest that the G and H frag- we cannot clearly detect the F-fragment in the ARGS- ments are further processed at a fast rate at a site near the Western [Fig. 3(B)] due to numerous ARGS-containing GELE epitope before further processing towards the G2 fragments detected with the BC3 antibody, presumably domain. The hypothesis that further proteolytic processing through further proteolytic cleavage of the fragments once of the fragments occurs in the culture media and not the they elute into the culture media. IL-1ra and anti-TNF- matrix is supported by the ﬁnding that we can only detect antibodies blocked the release of these fragments. one ARGS-containing fragment in the cartilage matrix, We next evaluated production of aggrecan fragments namely the 250 kDa H-fragment [see experiments below, resulting from cleavage at two other ADAM-TS4/TS5 Fig. 5(c)]. C-terminal cleavage sites, one between residues Glu1480 Finally, the culture media were monitored for 1872LGQR- and Gly1481 and the other between Glu1871 and Leu1872. containing products by Western analysis. The blot Using the anti-GELE polyclonal antibody the culture media showed that IL-1 and TNF increased the levels of a 98 kDa and cartilage extracts were analysed for GELE1481- LGQR-reactive fragment (I-fragment) and that this containing products. We detected a high MW fragment induction was blocked by IL-1ra and by the TNF antibody near 350 kDa in the cartilage extract [Fig. 4(b)] and a [Fig. 4(d)]. 546 M. D. Tortorella et al.: ADAM-TS4/TS5 in cartilage aggrecan breakdown

Table III Cytokine-induced degradation of aggrecan in bovine articular cartilage explants* Treatment GAG Levels (g/mg) Control 1.5±0.25 IL-1 [100 ng/ml] 14.4±3.40 IL-1+SE206 [10 M] 2.2±0.32 IL-1+XS309 [10 M] 15.2±4.40

*Bovine cartilage explants were incubated for 72 h with IL-1 and MMP-inhibitors, at the speciﬁed concentrations. At the end of the culture period, media were analysed for GAG content by DMMB assay. Mean values±S.D. of N=6 are shown.

Fig. 5. IL-1-induced degradation of aggrecan in bovine articular chelating the zinc atom in the catalytic domain required for cartilage explants. Bovine cartilage explants were incubated for enzymatic activity. XS309 is a nanomolar inhibitor of a large 2 h, 4 h, 6 h, 8 h, or 16 h with media containing IL-1 (100 ng/ml). At number of MMPs, but is inactive at >10M in blocking the end of the culture period, aggrecan and aggrecan catabolites ADAM-TS4/TS5 (designated ‘MMP-specific inhibitor’). In were extracted from the cartilage matrix and analysed for (a) the contrast, SE206 is a potent inhibitor of both MMPs and C-terminus KEEE1667, (b) the C-terminus GELE1480, or (c) the ADAM-TS4/TS5 (designated ‘general metalloproteinase 374 N-terminus ARGS by Western blot analysis on 4–12% gels inhibitor’). Explants were cultured for 72 h with IL-1 loaded on an equal GAG basis. (100 ng/ml), in the presence of either XS309 (10 M) or SE206 (10 M). Culture media were analysed for GAG-levels and culture media as well as cartilage matrix We also monitored the order in which aggrecan frag- extracts were analysed for KEEE1667 and GELE1480 ments were generated during IL-1 stimulated degradation. neoepitopes. It was found that the general metallo- We have found using soluble aggrecan and recombinant proteinase inhibitor, SE206, blocked IL-1-induced GAG ADAM-TS415 and ADAM-TS5 [Tortorella et al., in prep- release into the media (Table III), and the generation of the aration], that cleavage occurs preferentially at the Glu1667– KEEE1667 and GELE1480 neoepitopes in the media and Gly1668 site, then between Glu1480 and Gly1481 with the cartilage extracts [Fig. 6(a)–(d)]. In contrast, XS309 slowest rate at the Glu373–Ala374 site. In order to determine showed no inhibition of either GAG release or the formation whether the same order of cleavage was observed when of these neoepitopes. It is important to note that constitutive cartilage explants were incubated with IL-1, we evaluated levels of both KEEE1667- and GELE1480-containing frag- fragments within the cartilage matrix at early time points of ments were detected in the culture media of untreated incubation with IL-1. We looked in the matrix as opposed to explant cultures and the release of these fragments was the culture media, because it is only after 16 h that aggre- also blocked by the general metalloproteinase inhibitor, can fragments were detected in the culture media. In the SE206. This may suggest that either ADAM-TS4 and/or matrix, a 375 kDa KEEE1667 fragment was detected as ADAM-TS5 are involved in the constitutive turnover of early as 2 h after addition of IL-1 [Fig. 5(a)]. Next, the aggrecan, as well as the inducible breakdown. Interest- GELE1480 epitope was detected at 4 h (Fig. 5(b)], and the ingly, in the IL-1-stimulated calf articular cartilage the cul- 250 kDa 374ARGSV fragment was first detected after 8 h ture media contained two major KEEE-reactive fragments, [Fig. 5(c)]. Note the GELE-reactive fragment observed at the 300 kDa F-fragment and the 55 kDa J-fragment 4 h was a 350 kDa band corresponding to an aggrecan which is formed when the F-fragment is cleaved at the fragment with an intact G1 domain, generated by cleavage Glu1480–Gly1481 bond. The latter was not detected in the at the Glu1480–Gly1481 bond. It was followed at 8 h by the cultured media from IL-1 or TNF-stimulated cartilage from appearance of a 250 kDa GELE fragment with a 374ARGSV the older cow [Fig. 4(b)]. amino terminus, corresponding to the band seen in the BC-3 Western in Fig. 5(c), generated by cleavage of the 373 374 350 kDa fragment at the Glu –Ala bond. In conclu- INDUCTION OF ADAM-TS4 AND ADAM-TS5 sion, the fragmentation of aggrecan after stimulation of cartilage with IL-1 follows a similar temporal sequence to As the experiments described above suggest a role for that seen when soluble aggrecan is digested with recom- ADAM-TS4 and/or ADAM-TS5 in IL-1 and TNF-induced binant ADAM-TS4 and ADAM-TS5. aggrecan cleavage, we examined whether these cytokines triggered expression of ADAM-TS4 and ADAM-TS5 in bovine cartilage. We first analysed mRNA levels of INHIBITION OF CLEAVAGE BY A GENERAL METALLOPROTEINASE ADAM-TS4 and ADAM-TS5 by RT-PCR. Since we could INHIBITOR, BUT NOT AN MMP-SPECIFIC INHIBITOR not extract sufficient amounts of RNA from the explants to load equal amounts of RNA for the RT-PCR, the GAPDH In order to confirm that IL-1-induced breakdown of mRNA levels were determined for comparison [Fig. 7(a)]. aggrecan in explant cultures of bovine articular cartilage ADAM-TS4 message was not detected in control explants was mediated by ADAM-TS4 and ADAM-TS5, inhibition but it was strongly induced following IL-1 and TNF stimula- studies were performed using two hydroxamic acids tion and this induction was abrogated by IL-1ra or anti-TNF, which differ in their ability to inhibit the ADAM-TS enzymes. respectively [Fig. 7(b)]. In contrast, ADAM-TS5 mRNA These small molecules block metalloproteinases by was present in control explants, and the levels were not Osteoarthritis and Cartilage Vol. 9, No. 6 547 ab

Fig. 6. Blockage of IL-1-induced degradation of aggrecan in bovine articular cartilage explants. Bovine cartilage explants were incubated for 72 h with media containing IL-1 (100 ng/ml), with or without the MMP-speciﬁc inhibitor XS309 or the general metalloproteinase-inhibitor SE206. At the end of the culture period, culture media were analysed for (a) the C-terminus KEEE1667 and (b) the C-terminus GELE1480 by Western blot analysis on 4–12% gels loaded on an equal volume basis and the cartilage extracts were analysed for (c) KEEE1667 and (d) GELE1480 by Western blot analysis on 4–12% gels loaded on an equal GAG basis.

dramatically altered in the presence of the pro- bovine articular cartilage were immuno-adsorbed with an inflammatory cytokines [Fig. 7(c)]. In order to avoid the ADAM-TS4 or ADAM-TS5 affinity-purified polyclonal anti- variability due to the difficulty of RNA extraction from body coupled to protein-G Sepharose [Fig. 9(a)]. An cartilage matrix, we repeated the experiment using bovine ADAM-TS1 antibody coupled to protein G Sepharose was articular chondrocytes cultured in confluent monolayers, used as a negative control. Immunodepleted media were and again found that ADAM-TS5 message was expressed tested for aggrecanase activity by incubation with purified constitutively while ADAM-TS4 message was induced by aggrecan for 4 h at 37°C. Following the incubation, cleav- IL-1 and by TNF (data not shown). age of aggrecan at the Glu373–Ala374 site was monitored by We next examined ADAM-TS4 and ADAM-TS5 protein ELISA using the BC-3 monoclonal antibody. Media from levels produced by cartilage explants. Western Blot analy- untreated, control cartilage showed no detectable levels of sis of the culture medium of explants showed no ADAM- activity, whereas aggrecanase activity was detected in TS4 or ADAM-TS5 (not shown). Only guanidinium chloride media from IL-1-stimulated cartilage. Immunoprecipitation extracts from cartilage stimulated with IL-1 or TNF were ∼ positive for ADAM-TS4 (Fig. 8). The blot revealed three with the ADAM-TS4 antibody led to a 75% loss of aggre- bands with the approximate molecular weights of 60-, 45- canase activity in media from IL-1-stimulated cartilage. Immunoprecipitation with the ADAM-TS5 antibody led to a and 30-kDa. ADAM-TS5 was not detected under the con- ∼ ditions employed (not shown), which may be consistent 15% decrease in activity [Fig. 9(a)]. Finally, immuno- with it being constitutively expressed but only present at precipitation with the ADAM-TS1 antibody showed no very low levels, below the detection limit of the Western decrease in aggrecanase activity. The efficiency of Blot analysis. immunoprecipitation has been determined for each antibody. Both the ADAM-TS4 and ADAM-TS5 antisera successfully removed 100 ng of enzyme that was added to IMMUNODEPLETION OF ADAM-TS4 AND ADAM-TS5 1 ml of DMEM medium [Fig. 9(b)]. In order to determine the efficiency of the ADAM-TS1 immunoprecipitation, we In order to confirm that ADAM-TS4 and ADAM-TS5 are looked at removal of enzyme by Western Blot analysis responsible for the cytokine inducible aggrecan break- (data not shown). We achieved removal of >95% of all down, culture media from both control and IL-1-stimulated enzymes after immunoprecipitation. 548 M. D. Tortorella et al.: ADAM-TS4/TS5 in cartilage aggrecan breakdown a

Fig. 8. Effect of IL-1 and TNF on the expression of ADAM-TS4. Bovine cartilage explants were incubated for 72 h with (a) control media or in media containing either (b) IL-1 (100 ng/ml), (c) TNF (100 ng/ml), (d) IL-1 plus IL-1ra (10 g/ml), or (e) TNF plus neutralizing antibodies (10 g/ml). At the end of the culture period, proteins were extracted from the matrix and analysed for the presence ADAM-TS4 on 4–12% Tris-glycine gels loaded on an equal protein basis. c

domain of aggrecan, at the Glu373–Ala374 site19, we found that MT1-MMP cleaved native aggrecan at the MMP site between residues Ser341 and Phe342, but not at any of the aggrecanase sites, suggesting that the interaction of MT1-MMP with native aggrecan is different from rAgg1mut. We have shown that the temporal appearance of neoepitopes generated by cleavage at these sites in IL-1-treated cartilage corresponds with that seen upon cleavage of aggrecan by recombinant ADAM-TS4 and ADAM-TS5 (15 and Tortorella et al., in preparation). The Fig. 7. Effect of IL-1 and TNF on the expression of ADAM-TS4 and preferred site of cleavage of aggrecan by both ADAM-TS4 ADAM-TS5 mRNA. Cartilage explants were cultured for 72 hours and ADAM-TS5 is between residues Glu1667–Gly1668, in (a) control media, (b) IL-1 (100 ng/ml), (c) TNF (100 ng/ml), resulting in the generation of a 375 kDa fragment with an (d) IL-1 (100 ng/ml) plus IL-1ra (10 g/ml), and (e) TNF (100 ng/ml) intact N-terminus and a new C-terminus KEEE1667. Corre- plus anti-TNF (10 g/ml). At the end of the culture period, RNA was spondingly, the 375 kDa KEEE fragment was detected first extracted from the explants, measured and used for RT-PCR for in the matrix following IL-1 stimulation. Cleavage of aggre- (a) bovine GAPDH, (b) ADAM-TS4, yielding the predicted PCR 1480 product of 693 bp, (c) ADAM-TS5, yielding the predicted PCR can by ADAM-TS4/TS5 between residues Glu and 1481 product of 672 bp. Full length cDNA for ADAM-TS4 and ADAM- Gly is the next most favorable, resulting in the formation TS5 was included as a positive control. of a 350 kDa fragment with an intact N-terminus and a new C-terminus GELE1480. This is followed by cleavage within the IGD of aggrecan between residues Glu373 and Ala374, Discussion resulting in the formation of a 250 kDa fragment with a new N-terminus 374ARGS and the C-terminus GELE1480. It had previously been shown that aggrecan degradation The GELE-reactive fragment was the second fragment in bovine articular cartilage in response to IL-1 and TNF is detected in the cartilage matrix following IL-1 treatment. associated with the release of products generated by This fragment was first detected at 4 h, 2 h after the cleavage at the Glu373–Ala374 bond27. In the present KEEE-reactive fragment was detected. The ARGS-reactive studies, we demonstrate that aggrecan fragments resulting fragment was the last fragment to be detected in the matrix from cleavage at the ADAM-TS4/TS5 sensitive sites within following IL-1 stimulation (at about 8 h), consistent with the the C-terminus of aggrecan are also generated in cartilage kinetics of aggrecan cleavage by recombinant human explant cultures following IL-1 and TNF stimulation. Since ADAM-TS4/TS5. These data provide further evidence to we demonstrate that MMP-8 or MT1-MMP, two enzymes support ADAM-TS4 and/or ADAM-TS5 as the enzymes that have been reported to cleave at the Glu373-Ala374 responsible for cleavage in response to IL-1 and TNF. bond18,19, do not cleave at the ADAM-TS4/TS5-preferred The conclusion that ADAM-TS4 and/or ADAM-TS5 are sites, antibodies that recognize these neoepitopes are degrading aggrecan in this system is corroborated by the more specific for aggrecanase-activity. Unlike Buttner et al., finding that the general metalloproteinase inhibitor, SE206, who reported that MT1-MMP cleave rAgg1mut, a recom- blocked IL-1- and TNF-induced aggrecan breakdown, as binant substrate composed of the complete interglobular assessed by DMMB assay. This correlated with inhibition of Osteoarthritis and Cartilage Vol. 9, No. 6 549 a

Fig. 9. Immunodepletion of aggrecanase activity from culture media with ADAM-TS1, ADAM-TS4 or ADAM-TS5 antibodies. Two hundred l of culture media from bovine articular cartilage stimulated with IL-1 were immunodepleted with purified rabbit IgG, ADAM-TS1, ADAM-TS4 or ADAM-TS5 antibody-coupled Sepharose. Following depletion the media incubated with 500 nM of purified aggrecan for 4 h at 37°C and aggrecan products were analysed for the cleavage at the Glu373–Ala374site using the BC-3 monoclonal antibody in an ELISA. Non-immunodepleted culture media was also analysed to determine the maximum aggrecanase-activity present (a). To determine the efficiency of the immunoprecipitation using the ADAM-TS4 and ADAM-TS5 antibodies, 100 ng of the recombinant human enzyme was added to 1 ml of DMEM media. Following the addition of either ADAM-TS4 or ADAM-TS5 the enzymes were removed by immunoprecipitation as described above and the depleted medium was analysed for aggrecanase activity in the BC-3 ELISA (b). 550 M. D. Tortorella et al.: ADAM-TS4/TS5 in cartilage aggrecan breakdown the generation of the KEEE1667 and GELE1481 neoepitopes ADAM-TS4 and ADAM-TS5. Recently, Flannery et al. have in the culture media. The MMP-specific inhibitor, XS309, detected mRNA for ADAM-TS1 ADAM-TS2, ADAM-TS3, failed to inhibit aggrecan degradation, suggesting that ADAM-TS4, ADAM-TS5, and ADAM-TS6 in bovine carti- MMPs, such as MMP-8 and MT1-MMP which have been lage explants, bringing up the possibility that one of these shown to have the potential to cleave at the Glu373–Ala374 other ADAM-TS proteins may have aggrecanase activity31. aggrecanase site, are unlikely to play a major role in the It has been reported by Kuno et al. that ADAM-TS1 can breakdown of cartilage aggrecan. cleave cartilage aggrecan at the Glu1871–Leu1872 bond at a An important role for ADAM-TS4 and ADAM-TS5 in high concentration32. In our laboratory, we have shown that aggrecan cleavage is further supported by studies in which ADAM-TS1 can only cleave aggrecan at the Glu1871– immunodepletion with an ADAM-TS4 antibody led to a 75% Leu1872 bond at a 100-fold greater concentration than reduction and immunodepletion with an ADAM-TS5 anti- ADAM-TS4 or ADAM-TS5. At these high concentrations, body led to a 15% reduction of aggrecanase activity in the ADAM-TS1 was still not able to cleave aggrecan at the culture media of IL-1 stimulated bovine articular cartilage. other cleavage sites, between Glu373 and Ala374, Glu1480 Although a portion of the ADAM-TS4 and ADAM-TS5 and Gly1481, Glu1667 and Gly1668, and Glu1771 and Ala1772. protein may be retained in the cartilage matrix bound to For example, we found that as little as 500 pM of ADAM- aggrecan and other matrix proteins, we were unable to TS4 or ADAM-TS5 is needed to cleave aggrecan at all monitor the cartilage extracts for aggrecanase activity due these sites. In contrast, a concentration of 200 nM of to very high levels of aggrecan not found in the culture ADAM-TS1 was required to generate cleavage at the media and pre-existing ARGS, KEEE, GELE-reactive frag- Glu1871–Leu1872 bond (Tortorella et al., in preparation). ments that could not be removed by anion-exchange Based on the low specific activity of ADAM-TS1 for chromatography. Therefore, since we could not take into aggrecan, we feel that ADAM-TS1 is not a cartilage account any potential matrix-associated aggrecanase aggrecanase. activity, our data do not clearly define which is the more Aggrecan fragments with the amino terminal sequence important enzyme in this system. However, from these 374ARGS have been identified in synovial fluid of patients studies we can conclude that ADAM-TS-4 and ADAM-TS5 with osteoarthritis33 and inflammatory joint disease34. are responsible for approximately 90% of the aggrecanase Because MMP-8 and MT1-MMP are able to cleave at the activity found in culture media following IL-1 stimulation of Glu373–Ala374 bond to generate 374ARGS-containing frag- bovine articular cartilage, and ADAM-TS4 is responsible for ments18,19, this neoepitope may not be specific for the majority of this activity. aggrecanase-activity. Therefore, analysis of synovial fluid Both ADAM-TS4 and ADAM-TS5 were detected at the for the presence of neoepitopes generated by the message level in chondrocytes and cartilage after IL-1 and additional cleavage sites in the chondroitin sulfate-rich TNF stimulation. Interestingly, ADAM-TS5 message was region of aggrecan will enable us to further establish the detected in control cartilage, whereas ADAM-TS4 message role of these enzymes in human arthritis. Importantly, these was not. This suggests that ADAM-TS5 is constitutively neoepitope antibodies will also provide us with a means of expressed in cartilage, as has been proposed by others28, monitoring activity of ADAM-TS4 and ADAM-TS5 at early whereas ADAM-TS4 is induced. However, since we did not stages of disease, and thus may represent potential extract mRNA from cartilage immediately after dissection, markers of disease activity. we cannot rule out the possibility that ADAM-TS4 may be Although our data suggest a potential role for ADAM-TS4 switched off or ADAM-TS5 message is switched on during and/or ADAM-TS5 in arthritic disease, bovine cartilage preculture in the presence of FCS. explants in these studies were stimulated with 100 ng/ml ADAM-TS4 protein was detected in IL-1- or TNF-treated IL-1 or TNF. These levels may not be physiologically explants, but not in the control explants. However, this relevant and may represent a very aggressive induction of protein was not detected in the culture medium. Binding of cartilage breakdown, probably more aggressive than that ADAM-TS4 to the cell surface or to matrix components may occurring in chronic human arthritic disease. We do not explain this lack of detectable protein in the media. The know what the in vivo triggers are for aggrecanase induc- ADAM-TS proteins contain a thrombospondin type I motif tion, and studies suggest that IL-1 only induces minimal through which they have been shown to bind to the GAG aggrecan breakdown in human articular cartilage explant side chains of aggrecan29 and may bind to other matrix cultures (35 and Tortorella et al. unpublished data). There- components as well, or to the cell membrane as proposed fore, our future studies will focus on determining the role of previously30, either through this motif or via the disintegrin- ADAM-TS4/ADAM-TS5 in human articular cartilage aggre- like domain of the molecule. ADAM-TS5 protein was not can degradation and on what factors trigger the production detected in either the matrix or in the media. This may of these proteins. reflect low constitutive expression in cartilage that is not enhanced by cytokine stimulation. Because ADAM-TS4 is Acknowledgments regulated by IL-1 and TNF, it is interesting to speculate that this enzyme may be primarily responsible for the cytokine- The authors are grateful to the following scientists for induced aggrecan degradation that is observed in these their kind gifts; Dr Jerry Saklatvala (Kennedy Institute of studies. However, further work is needed before any Rheumatology, London, U.K.) for the IL-1 and IL-1 recep- definitive conclusions can be made. tor antagonist (IL-1ra), Professor W. Stec (Centre of In summary, our studies suggest that ADAM-TS4 and/or Molecular and Macromolecular Biology, Lodz, Poland) for ADAM-TS5 represent the endogenous aggrecanases the TNF, and Dr Amanda Fosang (University of Melbourne, responsible for a large part of the aggrecan breakdown in Parkville, Australia) for the AF-28 antibody. bovine cartilage explants treated with IL-1 or TNF. This could mean that they are also the enzymes responsible for References aggrecan breakdown in human disease. However, this does not rule out the involvement of other proteinases yet 1. Paulsson M, Morgolin M, Wiedemann H, Beardmore- to be discovered that have similar substrate specificity to Gray M, Dunham D, Hardingham TE, et al. Extended Osteoarthritis and Cartilage Vol. 9, No. 6 551

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