Distinguishing Aggrecan Loss from Aggrecan Proteolysis in ADAMTS-4 and ADAMTS-5 Single and Double Deficient Mice

Abstract

Aggrecan loss from mouse cartilage is predominantly because of ADAMTS-5 activity; however, the relative contribution of other proteolytic and nonproteolytic processes to this loss is not clear. This is the first study to compare aggrecan loss with aggrecan processing in mice with single and double deletions of ADAMTS-4 and -5 activity (⌬cat). Cartilage explants harvested from single and double ADAMTS-4 and -5 ⌬cat mice were cultured with or without interleukin (IL)-1␣ or retinoic acid and analyzed for (i) the kinetics of 35 S-labeled aggrecan loss, (ii) the pattern of 35 S-labeled aggrecan fragments released into the media and retained in the matrix, (iii) the pattern of total aggrecan fragments released into the media and retained in the matrix, and (iv) specific cleavage sites within the interglobular and chondroitin sulfate-2 domains. The loss of radiolabeled aggrecan from ADAMTS-4/-5 ⌬cat cartilage was less than that from ADAMTS-4, ADAMTS-5, or wild-type cartilage under nonstimulated conditions. IL-1␣ and retinoic acid stimulated radiolabeled aggrecan loss from wild-type and ADAMTS-4 ⌬cat cartilage, but there was little effect on ADAMTS-5 cartilage. Proteolysis of aggrecan contributed most to its loss in wild-type, ADAMTS-4, and ADAMTS-5 ⌬cat cartilage explants. The pattern of proteolytic processing of aggrecan in these cultures was consistent with that occurring in cartilage pathologies. Retinoic acid, but not IL-1␣, stimulated radiolabeled aggrecan loss from ADAMTS-4/-5 ⌬cat cartilage explants. Even though there was a 300% increase in aggrecan loss from ADAMTS-4/-5 ⌬cat cartilage stimulated with retinoic acid, the loss was not associated with aggrecanase cleavage but with the release of predominantly intact aggrecan consistent with the phenotype of the ADAMTS-4/-5 ⌬cat mouse. Our results show that chondrocytes have additional mechanism for the turnover of aggrecan and that when proteolytic mechanisms are blocked by ablation of aggrecanase activity, nonproteolytic mechanisms compensate to maintain cartilage homeostasis.Aggrecan catabolism has been studied extensively in the past 30 -40 years as part of the metabolic processes important in normal functional cartilage and in cartilage pathology. The major enzymes involved in degradation of aggrecan in normal and pathological cartilage are aggrecanases members of the ADAMTS 2 subfamily of adamalysin (M12) metalloproteinases (1, 2). These enzymes cleave the aggrecan core protein at aggrecanase-specific cleavage sites between glutamate and a small hydrophobic amino acid residue (3, 4). Most studies suggest that in vitro and in vivo ADAMTS-4 and -5 are the main aggrecanases (5-11); however, ADAMTS-1, -8, -9, -15, -16, and -18 also exhibit aggrecanase activity (12-15). Recombinant ADAMTS-4 and -5 have been suggested to be the most potent aggrecanases (15-19), although the relative potency in aggrecan degrading activity has yet to be clarified (11,20,21). In addition, recombinant ADAMTS-1, -4, and -5 cleave aggrecan at 5 sites: one within the interglobular domain a...

ADAMTS-5: The story so far

“…Alternatively, glycosidases such as hyaluronidase could have more prominent roles in resorbing the aggrecan aggregate in growth cartilage (Durigova et al, 2007;Sztrolovics et al, 2002). The latter hypothesis is supported by data showing a lack of skeletal abnormality in ADAMTS-4-deficient mice (Glasson et al, 2004;Stanton et al, 2005), ADAMTS-5-deficient mice (Glasson et al, 2005;Stanton et al, 2005), ADAMTS-4/-5 doubledeficient mice (Ilic et al, 2007;Majumdar et al, 2007;Rogerson et al, 2008), and aggrecan knockin mice resistant to aggrecanase cleavage in the IGD . Aggrecan knockin mice resistant to MMP cleavage in the IGD are also skeletally normal .…”

“…Adenovirus infection of mouse synovium with connective tissue growth factor induces ADAMTS-5 expression and synovial fibrosis (Blaney Davidson et al, 2006). Galectin-3, a soluble lectin increased in human OA cartilage (Guevremont et al, 2004), weakly stimulates ADAMTS-5 expression in human OA chondrocytes (Janelle-Montcalm et al, 2007). Conversely, nutraceuticals such as glucosamine and chondroitin sulphate at biologically relevant concentrations decrease IL-1-induced ADAMTS-5 expression in bovine cartilage explants (Chan et al, 2006).…”

“…Identifying the enzyme families responsible for creating the fragments described above is not straightforward because there are exceptions to the rules for aggrecan neoepitope specificity. At high concentrations in vitro, MMP-8 (Fosang et al, 1994) and MMP-14 (Büttner et al, 1998) cleave at the E 373 374 A aggrecanase site, and ADAMTS-1 (RodriguezManzaneque et al, 2002), ADAMTS-4 and cathepsin B [East et al, 2007] consistent with a number of studies reporting immunodetection of DIPEN 341 (or DIPES 341 in the bovine) neoepitope without detection of the corresponding 342 FFGVG neoepitope ADAMTS-5: The story so far epitope could either be due to an absence of aggrecanolysis (the fragment is not formed), or abundant aggrecanolysis which converts the entire fragment to SELE 1279 (see Fig 2). Thus, more than one neoepitope is needed to characterise the full extent of aggrecanolysis.…”

Chondropenia: current concept review

“…The aggrecanase could pave the way for new therapeutic strategies, since it has been observed that ADAMTS-4 and ADAMTS-5 gene deletion decreases the severity of OA in animal models [77,78]. The TIMP-3 (tissue inhibitor of metalloproteinases-3) is the most important endogenous aggrecanase inhibitor identified so far [36,79].…”

Emerging roles for ADAMTS5 during development and disease

“…All members of the ADAMTS proteoglycanases have been shown to, or are predicted to, cleave aggrecan however ADAMTS5 has emerged as the most important "aggrecanase" (pathologically), at least in the mouse, whereby catalytically inactivated Adamts5 (ΔCAT) homozygous allelic mice are protected from experimentally induced arthritis (Glasson et al, 2005;Stanton et al, 2005), which is not the case for Adamts1 and Adamts4 (Ilic et al, 2007) homozygous knockout mice. Most members of the ADAMTS proteoglycanases have also been shown to, or are predicted to, cleave versican (Sandy et al, 2001;Russell et al, 2003;Somerville et al, 2003;Westling et al, 2004;Silver et al, 2008;Longpre et al, 2009;McCulloch et al, 2009b) (Table 1) with ADAMTS5 showing the highest activity of all ADAMTS proteoglycanases characterised to date toward versican in vitro at the G1-DPEAAE cleavage site (pers.…”