ADAMTS‐4 and ADAMTS‐5 sequestration and activity in chondrocyte‐agarose cultures

Introduction A primary event in the destruction of cartilage in arthritic diseases is the loss of aggrecan from the extracellular matrix of articular cartilage. During aggrecan breakdown, cleavage sites are utilized, which reside within the IGD of the aggrecan core protein. The Asn 341 –Phe 342 bond is cleaved by members of the MMP family, whereas the second of the two cleavage sites, the Glu 373 –Ala 374 bond, is cleaved by the aggrecanases which are all members of the A disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS) family. Both ADAMTS‐4 and ‐5 have been shown to readily cleave aggrecan at this so‐called aggrecanase site ( Tortorella et al . 1999 ; Abbaszade et al . 1999 ; Sandy et al . 2000 ). An in vitro model of cartilage degradation has also shown that these enzymes are responsible for the loss of aggrecan from explant cultures of articular cartilage stimulated with IL‐1‐a or TNF‐a ( Tortorella et al . 2001 ). Both ADAMTS‐4 and ‐5 are thought to be synthesized as inactive zymogens that are activated via removal of their propeptide domain by the Golgi enzyme furin. The secreted active ADAMTS‐4 and ‐5 have predicted molecular weights of 67.9 and 73.6 kDa, respectively. The objective of this study was to monitor ADAMTS‐4 and ‐5 secretion and sequestration in the extracellular matrix of chondrocyte‐agarose cultures. Methods Porcine articular chondrocytes were isolated and embedded in agarose ( Hughes et al . 1997 ) before preculture in DMEM + 50 µg/ml gent. with 10% FBS and 25 µg/ml Phos.C for 21 days. The plates were washed, then cultured in serum‐free DMEM with or without IL‐1‐α for 96 h. GAG release to the medium was measured using the DMMB assay. Media samples were analysed by Western blotting for aggrecan metabolites using mAb BC‐3 to recognize the aggrecanase‐generated neoepitope ARGSV. The presence of ADAMTS‐4 in the media was analysed using the mAb anti‐TS‐4N, which recognizes the metalloproteinase domain, and commercially available polyclonal antibodies to the pro‐ and spacer domains of ADAMTS‐4. The presence of ADAMTS‐5 was detected using commercially available polyclonal antibodies to the pro‐ and spacer domains of ADAMTS‐5. Agarose plugs were extracted in detergent buffer and analysed by Western blotting for ADAMTS‐4 and ‐5 using the same monoclonal and polyclonal antibodies. Results In control cultures, only 20–30% of the total GAG was released into the medium after 96 h of culture. In contrast, 80–90% of the total GAG was released in cultures exposed to IL‐1. Western blot analysis showed aggrecanase‐generated aggrecan metabolites in the IL‐1‐treated cultures but none in the control cultures. Sequestered forms of both ADAMTS‐4 and ‐5 are present in the matrix prior to treatment in serum‐free conditions, and following treatment with or without IL‐1 for 96 h, there are no differences in the high molecular weight isoforms of the enzymes sequestered in the matrix. Western blots of partially purified media samples showed no differences in the zinc chelator‐bound isoforms of either ADAMTS‐4 or ‐5 between control and IL‐1‐treated cultures. However, the predominant heparin sepharose‐bound isoforms of ADAMTS‐4 and ‐5 co‐migrate at approximately 37 kDa. Each of the heparin‐bound 37‐kDa isoforms of ADAMTS‐4 and ‐5 are detected in increased amounts in IL‐1α‐treated cultures compared to controls. Discussion The increased amounts of the 37‐kDa isoforms of both ADAMTS‐4 and ‐5 in the IL‐1‐treated cultures suggest a role for these smaller isoforms in the increased aggrecanase activity seen in the IL‐1‐treated cultures compared to controls. This study has identified multiple isoforms of putative aggrecanase activity that could be responsible for increased aggrecan catabolism that leads to cartilage degradation in arthritis.