Aggrecanase versus matrix metalloproteinases in the catabolism of the interglobular domain of aggrecan in vitro

Abstract

The importance of aggrecanase versus matrix metalloproteinase (MMP) enzymic activities in the degradation of aggrecan in normal and osteoarthritic (OA) articular cartilage in vitro was studied in order to further our understanding of the potential role of these two enzyme activities in aggrecan catabolism during the pathogenesis of cartilage degeneration. Porcine and bovine articular cartilage was maintained in explant culture for up to 20 days in the presence or absence of the catabolic stimuli retinoic acid, interleukin-1 or tumour necrosis factor-α. Release of proteoglycan from cartilage was measured as glycosaminoglycan (GAG) release using a colorimetric assay. Analysis of proteoglycan degradation products, both released into culture media and retained within the cartilage matrix, was performed by Western blotting using antibodies specific for the N- and C-terminal neoepitopes generated by aggrecanase- and MMP-related catabolism of the interglobular domain of the aggrecan core protein (IGD). In addition, studies determining the mRNA expression for MMP-3 and MMP-13 in these same cultures were undertaken. These analyses indicated that all three catabolic agents stimulated the release of > 80% of the GAG from the articular cartilage over 4 days. The degree of GAG release corresponded to an increase in aggrecanase-generated aggrecan catabolites released into the media and retained within the cartilage. Importantly, there was no evidence for the release of MMP-generated aggrecan metabolites into the medium, nor the accumulation of MMP-generated catabolites within the tissue in these same cultures. Expression of the mRNAs for two MMPs known to be capable of degrading the aggrecan IGD, MMP-3 and MMP-13, was detected. However, increased expression of these MMPs was not correlated with aggrecan degradation. Analyses using porcine cartilage, cultured with or without catabolic stimulation for 12 h to 20 days, indicated that primary cleavage of the IGD by aggrecanase was responsible for release of aggrecan metabolites at both the early and late time points of culture. Cultures of late-stage OA human articular cartilage samples indicated that aggrecanase activity was upregulated in the absence of catabolic stimulation when compared with normal porcine or bovine cartilage. In addition, even in this late-stage degenerate cartilage, aggrecanase and not MMP activity was responsible for the release of the majority of aggrecan from the cartilage. This study demonstrates that the release of aggrecan from both normal and OA cartilage in response to catabolic stimulation in vitro involves a primary cleavage by aggrecanase and not MMPs.

ADAMTS4 Cleaves at the Aggrecanase Site (Glu373-Ala374) and Secondarily at the Matrix Metalloproteinase Site (Asn341-Phe342) in the Aggrecan Interglobular Domain

“…Although it has been widely accepted (22,23,30,(37)(38)(39)(40)(41)(42) that ADAMTS4 (aggrecanase-1) cleaves only at the Glu 373 -Ala 374 bond within the IGD, the results described here suggest that both of the major cleavages that are known to occur in vivo, at Glu 373 -Ala 374 and Asn 341 -Phe 342 , can be catalyzed by ADAMTS4. Previously, cleavage at the Asn 341 -Phe 342 site in the aggrecan IGD was considered a specific property of the MMP family, with the exception of a snake venom enzyme (30,34) 341 -Phe 342 bond in the experiments described here was due to contamination of the ADAMTS4 preparation with an MMP-like activity was eliminated by the observation that although 10 mM EDTA and TIMP-3 totally blocked the cleavage, neither TIMP-1 nor TIMP-2 was inhibitory, even at 750 nM.…”

Generation and Novel Distribution of Matrix Metalloproteinase-derived Aggrecan Fragments in Porcine Cartilage Explants

“…Aggrecanase-derived 374 ARGSV fragments are the major aggrecan products found in synovial fluids from arthritis patients (7,8). Furthermore, the aggrecanase-derived ITEGE 373 neoepitope at the C terminus of the G1 domain has been detected in human (9,10), bovine (11,12), pig (12), and rat (11) cartilage and in mice with experimental arthritis (13,14).…”

“…The 342 FFGVG N-terminal neoepitope (Fig. 1c) has been found in human synovial fluids (16) and conditioned medium from porcine (22) and human osteoarthritic (12) cartilage cultures. The C-terminal neoepitope DIPEN 341 ( Fig.…”

Generation of Anti-complement “Prodrugs”

“…Cleavage of aggrecan is enhanced in inflammatory joint disease resulting in cartilage damage, indeed soluble products of aggrecan catabolism generated by aggrecanases and MMPs can be found in synovial fluid and cartilage extracts of patients with arthritis (14, 20 -22). In vitro culture systems have also demonstrated that a variety of inflammatory cytokines, such as IL-1␣ and TNF-␣ stimulate cleavage at these same sites (23)(24)(25)(26). The enzyme cleavage sites, and the minimal sequence lengths for recognition by target enzymes, have been previously documented (27)(28)(29)(30).…”

“…All of these reagents are known stimulators of cartilage catabolism. IL-1␣ and TNF-␣ are known to up-regulate the production of the aggrecanases and some MMPs, whereas retinoic acid is known to up-regulate the production of the aggrecanases but not the MMPs (23)(24)(25)(26)40). Following incubation of chondrocytes with these reagents and the DAF4-IGD75-IgG 4 prodrug, the neoepitopes generated by the aggrecanases and MMPs were detected using BC-3 and This generated a reagent with little Cregulatory activity until cleaved by MMPs at the site of inflammation, resulting in release of the active DAF moiety (C).…”