Cartilage chondrolysis by fibronectin fragments causes cleavage of aggrecan at the same site as found in osteoarthritic cartilage

FIBRONECTIN proteolytic fragments (Fn-fs) have very potent cartilage chondrolytic activities toward cultured cartilage explants in vitro [1] and when injected into rabbit knee joints [2]. Their relevance to cartilage pathology is supported by their presence at elevated levels in synovial fluids of patients with osteoarthritis and rheumatoid arthritis [3, 4]. Their relevance is also supported by the discovery that their effects are mediated through other factors, thought to have roles in cartilage pathology, catabolic cytokines. Cytokines have been detected in conditioned media of Fn- f treated human cartilage [5] and neutralization with antibodies directed toward specific cytokines blocks cartilage damage by Fn-fs and induction of matrix metalloproteinases (MMPs) [5]. The major MMP responsible for cartilage damage appears to be MMP-3 (stromelysin-1), since antibodies to MMP-3 greatly slow cartilage damage caused by the Fn-fs [6, 7] and conditioned media of Fn- f treated cartilage contains levels of up to 800 nM of active MMP-3 [7]. Since MMPs cleave at the the Asn360-Phe361 bond* in the interglobular domain of aggrecan [8], it would be expected that this cleavage would occur in Fn-f treated cartilage as well. However, this site is not cleaved in normal cartilage aggrecan turnover in vitro and in vivo [9] or in adult cartilage explants treated with interleukin-1 (IL-1) or retinoic acid [9- 11]. In the latter cases, cleavage occurs exclusively at Glu392-Ala393. Further, the expected N-terminal Ala aggrecan fragments are found in human synovial fluids of patients with both osteoarthritis [11] and inflammatory joint diseases [12]. Accordingly, the enzymatic activity that may be responsible for this has been called 'aggrecanase', but the responsible proteinase has not yet been identified or isolated. Our objective here was to determine whether the Fn-fs caused cleavage at the physiologically relevant 'aggrecanase' site or at the MMP site in bovine cartilage aggrecan. The use of bovine cartilage was appropriate since proteoglycan synthesis suppression, cartilage proteoglycan degradation and release of active MMP-3 occurs to similar extents in both bovine and human knee cartilage explants [5-7]. We report here that cleavage was detected at the aggrecanase site, as well as other sites, but not at the MMP site. Thus, these data support a potential physiological role for Fn-fs in cartilage pathology in osteoarthritis or inflammatory joint disease. The data also support the relevance of the use of Fn-fs in animal models of knee joint damage as described by us earlier [2] as well as in in vitro explant culture models [1, 5-7].