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CARTILAGE MECHANICAL INJURY AND CO-CULTURE WITH JOINT CAPSULE TISSUE INCREASE ABUNDANCE OF ADAMTS-5 PROTEIN AND AGGRECAN G1-NITEGE PRODUCT +*Lee, J H; **Bai, Y; §Flannery, CR; **Sandy, JD; **Plaas, A; *Grodzinsky, AJ *MIT, Cambridge, MA; §Wyeth Research, Cambridge, MA; **USF, Tampa, FL [email protected] INTRODUCTION: Joint injury in young adults leads to increased risk for development of osteoarthritis later in life [1]. Mechanical overload of cartilage explants [2] and co-culture with joint capsule tissue [3] have been established as in vitro models for the study of joint injury with the goal of elucidating processes of matrix degradation relevant to disease progression. These models of joint injury have been shown previously to increase gene expression of matrix degrading enzymes without affecting expression of matrix proteins [4,5]. The objectives of this study were to (1) determine if protein levels of aggrecanase-1 (ADAMTS-4) and aggrecanase-2 (ADAMTS-5) increase in response to mechanical injury of cartilage or co-culture with joint capsule tissue, and (2) determine if aggrecan is cleaved by aggrecanase activity in these injury models. METHODS: Tissue Harvest & Injury Models: Cartilage disks (3 mm diam, 1 mm think) from the middle zone of the femoropatellar groove and pieces of joint capsule tissue (~5 mm diam) were harvested from knee joints of 1-2 week old calves and maintained separately in medium (DMEM + 10% FBS) for two days to allow equilibration. On day 2, location-matched cartilage disks were either maintained in free swelling culture, mechanically injured (50% strain compression ramp at 1mm/s followed by immediate release), co-cultured with joint capsule tissue, or both injured and co-cultured. Immunohistochemistry: On days 4 and 16, cartilage and joint capsule tissue samples were fixed in formalin, paraffin embedded, sectioned, stained with anti-ADAMTS-4 (JSCVMA) or anti-ADAMTS-5 (JSCKNG) antibodies, and counterstained with Methylgreen. Proteinase K treatment was used for epitope retrieval. Aggrecan Western Blotting: On day 16, aggrecan was extracted from cartilage disks in 4M GuHCl + proteinase inhibitors followed by ethanol precipitation and digestion of GAGs (chondroitinase ABC, keratanase II, endo-β-galactosidase). Reduced samples were run on 4-15 % gradient Tris-HCl gels, transferred to nitrocellulose, and blotted with anti-G1 antibody or anti-NITEGE antibody (detects neo-epitope resulting from aggrecanase cleaveage at Glu 373 of aggrecan core protein). Antibody binding was detected by chemiluminescence. RESULTS: Aggrecanase immunostaining: Increased abundance of ADAMTS-5 was observed in cartilage tissue explants 4 days after injurious mechanical compression and following 4 days of co-culture with joint capsule tissue (Fig. 1). Increased staining for ADAMTS-5 continued through 16 days following injury and following injury + coculture; levels returned to control by day 16 in cartilage co-cultured with joint capsule but not injured (Fig. 1). Staining predominantly appeared to be associated with chondrocytes rather than in the extracellular matrix. Staining for ADAMTS-4 was low or absent at all time points with no discernible difference seen between experimental conditions (data not shown). Cleavage of Aggrecan: Accumulation of aggrecanasegenerated cleavage products in cartilage explants was observed following injury, co-culture with joint capsule, and the combination of injury + co-culture (Fig. 2). The cleavage site was confirmed using an antibody specific to the neo-epitope generated by cleavage at the Glu373Ala374 bond in the aggrecan core protein (data not shown). Analysis of conditioned culture medium for aggrecan fragments by Western blotting was complicated by release of G1 and NITEGE reactive proteins from the joint capsule tissue when cultured alone (data not shown). This release from bovine joint capsule alone, which is consistent with observations of other investigators [6], made analysis of the co-culture samples problematic in determining the source (cartilage or joint capsule) of proteins in the culture medium. No increase was observed in cleavage of aggrecan at the MMP site (VDIPES) in the IGD following injury or co-culture through 16 days in culture (data not shown). DISCUSSION: Mechanical injury and co-culture with joint capsule tissue resulted in increased abundance of ADAMTS-5 in cartilage explants and cleavage at the aggrecanase site in the interglobular domain (IGD) of aggrecan. In addition, we observed previously that gene expression of ADAMTS-5 increased 40-fold while ADAMTS-4 increased only 2-3-fold following injury alone [4]. During co-culture, gene expression of ADAMTS-4 and ADAMTS-5 were both upregulated ~6-fold [4,5]. ADAMTS-5 protein is also present in human arthritic synovial tissue [7] and was recently shown to be the primary aggrecanase responsible for cartilage degradation in two mouse models of arthritis [8,9]. The present data supports the idea that ADAMTS-5 (and not ADAMTS-4) is also responsible for aggrecanolysis in these bovine cartilage injury models. This appears to contrast with the finding that ADAMTS-4 protein and MT4MMP are increased in abundance and appear to be responsible for aggrecanolysis in bovine cartilage explants treated with interleukin-1 [10]. Ongoing analyses of both tissue and medium compartments with multiple quantitative assays (ELISA), determination of the activation status of the proteinases present, and siRNA strategies are focused on identifying definitively the relative importance of the different aggrecanases (ADAMTS-1,4,5,8,9,15) in cartilage degradation in different species and different biological environments. day 4 day 16 Control Injury Co-culture Injury + Co-culture Figure 1: Cartilage injury and co-culture with joint capsule result in an increase in ADAMTS-5 in the tissue. Control Injury Co-culture Inj+Co-cult 80 kD 60 kD Figure 2: Cartilage injury and co-culture with joint capsule cause accumulation of aggrecanase generated aggrecan fragments in the tissue stained with an antibody to the G1 domain of aggrecan. REFERENCES: [1] Gelber+, Ann Intern Med 133:3211-8, 2000. [2] Chen+, JOR 17:870-9. 1999. [3] Jubb+, Arth and Rheum 23:545-55, 1980. [4] Lee+, Arth and Rheum 52:2386-95, 2005. [5] Lee+, Trans ORS 51st #1032, 2005. [6] Yutani+, J Bone Miner Metab 17:7-10, 1999. [7] Vankemmelbeke+, Eur J Biochem 268:1259-69, 2001. [8] Glasson+, Nature 434:644-648, 2005. [9] Stanton+, Nature 434:648-652, 2005. [10] Patwari+, Osteoarth and Cart 13:269-77, 2005. 52nd Annual Meeting of the Orthopaedic Research Society Paper No: 0214