Expression and activity of ADAMTS‐5 in synovium

Abstract

ADAMTS proteinases, belonging to the adamalysin subfamily of metalloproteinases, have been implicated in a variety of cellular events such as morphogenesis, cell migration, angiogenesis, ovulation and extracellular matrix breakdown. Aggrecanase-1 (ADAMTS-4) and aggrecanase-2 (ADAMTS-5) have been identified in cartilage and are largely responsible for cartilage aggrecan breakdown. We have shown previously that synovium, the membrane lining diarthrodial joints, generates soluble aggrecanase activity. We report here the expression, localization and activity of ADAMTS-5 from human arthritic and bovine synovium. ADAMTS-5 was expressed constitutively in synovium with little or no transcriptional regulation by recombinant human interleukin-1a or all-trans-retinoate, factors previously shown to upregulate aggrecanase activity in cartilage. Aggrecanase activity generated by synovium in vitro and recombinant ADAMTS-5 cleaved aggrecan extensively, resulting in aggrecan fragments similar to those generated by chondrocyte-derived aggrecanases, and the activity was inhibited by heparin. ADAMTS-5 was immunolocalized in human arthritic synovium, where staining was mostly pericellular, particularly in the synovial lining and around blood vessels; some matrix staining was also seen. The possibility that synovium-derived ADAMTS-5 may play a role in cartilage aggrecan breakdown is discussed.Keywords: ADAMTS; aggrecanase; arthritis; synovium.The ADAMTS (a disintegrin and metalloproteinase with thrombospondin motifs) proteinases are a growing group of recently identified metallopeptidases related to the ADAMs (a disintegrin and metalloproteinase) [1]. They are classified in the Merops database of peptidases as members of subfamily B (the adamalysin subfamily) of family M12 in clan MA [2]. So far, nine mammalian ADAMTS proteinases have been identified [3]. They, unlike most ADAMs, do not possess an epidermal growth factor repeat domain or transmembrane domain but instead have a thrombospondin type I motif, a cysteine-rich spacer region and usually one or more C-terminal thrombospondin submotifs [4]. The thrombospondin type I repeats in thrombospondins 1 and 2 possess affinity for heparin and other sulfated glycosaminoglycans (sGAGs) as well as fibronectin and also contain cell adhesion sequences [5±7]. ADAMTS enzymes are therefore soluble proteins that may become incorporated into the extracellular matrix (ECM) after secretion, as is the case for ADAMTS-1 [8]. They may be activated during secretion through furin cleavage and release of the propeptide [9]. ADAMTS proteinases have been implicated in a wide range of ECM-degrading events [1,10±17]. For many of the ADAMTS proteinases however, biological functions remain to be assigned [3,18]. Two members, ADAMTS-1 and -8 have also been shown to possess potent antiangiogenic properties [19].Aggrecan, a large aggregating proteoglycan, is together with type II collagen the major constituent of articular cartilage. The type II collagen fibres provide the cartilage with tensile strength and agg...

Selective inhibition of ADAMTS‐1, ‐4 and ‐5 by catechin gallate esters

“…3) showed that they resulted from typical aggrecanase cleavages in poorly glycosylated regions of the aggrecan core protein. These cleavage sites have been reported for both ADAMTS-4 and -5 [17,31,42]. We describe here for the first time the N-terminal sequences of the major aggrecan fragments generated by ADAMTS-1.…”

“…ADAMTS-5 purified from cartilage-conditioned medium has revealed multiple bands in the range of 40-65 kDa [17]. In addition, similarly processed forms of the enzyme have been detected in synovium-conditioned medium from arthritis patients [31] and in a GnHCl extract from cartilage of an arthritic patient [23].…”

“…Aggrecan, purified from bovine nasal cartilage by extraction with 4 M guanidinium (Gn) HCl and dissociative CsCl 2 density gradient ultracentrifugation [29] was entrapped in polyacrylamide and used as a substrate to determine aggrecan-degrading activity as previously described [29][30][31]. Aliquots of the aggrecan/polyacrylamide particles (4 mg) were incubated with the respective enzymes in a total volume of 500 lL (assay buffer: 0.1 M Tris/HCl, 0.1 M NaCl, 10 mM CaCl 2 , 0.1% w/v Chaps, pH 7.5).…”

“…Briefly, 10 units of rhADAMTS-1, and 5 units of rhADAMTS-4 or -5 were incubated with 5 mg purified aggrecan monomer (see above) in enzyme assay buffer: 50 mM Tris/HCl, 0.1 M NaCl, 10 mM CaCl 2 , 0.1% w/v Chaps, pH 7.5 at 37°C for 16 h. Aggrecan fragments were detected by Western blotting using the monoclonal antibody 5/6/3-B-3 (ICN Flow) which recognizes terminal unsaturated chondroitin 6-sulfate disaccharides. The fragments were then isolated and subjected to N-terminal sequence analysis as previously described [31]. An additional control consisted of analysing the final enzyme preparations for potential contaminating MMP activity using a quenched fluorescence substrate which is cleaved by all MMPs, but which is not cleaved by aggrecanases (see below).…”

The role of proteases in pathologies of the synovial joint

“…In rheumatoid arthritis synovitis is associated with hyperplasia of the synoviocytes, an infiltration of mononuclear cells including B cells, T cells and macrophages into the synovial sublining, and the formation of a locally invasive pannus (Lee & Weinblatt, 2001). A number of studies have shown that in arthritic pathologies the synovial expression of a number of proteases, including MMP collagenases, ADAMTS aggrecanases and MMP-3, is increased as are the levels of these proteases in the synovial fluid (Hulejová et al, 2007;Smeets et al, 2003;Tchetverikov et al, 2004;Vankemmelbeke et al, 2001;Vankemmelbeke, Ilic, Handley, Knight, & Buttle, 1999). In vitro studies have implicated synovium-derived factors, in particular proinflammatory cytokines (Fell & Jubb, 1977;Saklatvala, Pilsworth, Sarsfield, Gavrilovic, & Heath, 1984) and proteases (Vankemmelbeke et al, 1999(Vankemmelbeke et al, , 2001) as mediators of cartilage destruction.…”