Heather Stanton

ADAMTS5 is the major aggrecanase in mouse cartilage in vivo and in vitro.

Aggrecan is the major proteoglycan in cartilage, endowing this tissue with the unique capacity to bear load and resist compression. In arthritic cartilage, aggrecan is degraded by one or more ‘aggrecanases’ from the ADAMTS (a disintegrin and metalloproteinase with thrombospondin motifs) family of proteinases. ADAMTS1, 8 and 9 have weak aggrecan-degrading activity. However, they are not thought to be the primary aggrecanases because ADAMTS1 null mice are not protected from experimental arthritis, and cleavage by ADAMTS8 and 9 is highly inefficient. Although ADAMTS4 and 5 are expressed in joint tissues, and are known to be efficient aggrecanases in vitro, the exact contribution of these two enzymes to cartilage pathology is unknown. Here we show that ADAMTS5 is the major aggrecanase in mouse cartilage, both in vitro and in a mouse model of inflammatory arthritis. Our data suggest that ADAMTS5 may be a suitable target for the development of new drugs designed to inhibit cartilage destruction in arthritis, although further work will be required to determine whether ADAMTS5 is also the major aggrecanase in human arthritis.

Proteoglycan degradation by the ADAMTS family of proteinases

Proteoglycans are key components of extracellular matrices, providing structural support as well as influencing cellular behaviour in physiological and pathological processes. The diversity of proteoglycan function reported in the literature is equally matched by diversity in proteoglycan structure. Members of the ADAMTS (A Disintegrin And Metalloproteinase with ThromboSpondin motifs) family of enzymes degrade proteoglycans and thereby have the potential to alter tissue architecture and regulate cellular function. In this review, we focus on ADAMTS enzymes that degrade the lectican and small leucine-rich repeat families of proteoglycans. We discuss the known ADAMTS cleavage sites and the consequences of cleavage at these sites. We illustrate our discussion with examples from the literature in which ADAMTS proteolysis of proteoglycans makes profound changes to tissue function.

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

We have studied aggrecan catabolism mediated by matrix metalloproteinases (MMPs) in a porcine cartilage culture system. Using antibodies specific for DIPEN341 and342FFGVG neoepitopes, we have detected MMP-derived fragments in conditioned medium and cultured cartilage, by radioimmunoassay, Western blotting, and immunolocalization. Radioimmunoassay revealed that the amount (pmol of epitope/mg of total glycosaminoglycan) of 342FFGVG epitope released from cartilage remained constant over a 5-day culture period and was not increased by IL-1α or retinoate. However, theproportion (pmol of epitope/mg of released glycosaminoglycan) of 342FFGVG epitope released was decreased upon stimulation, consistent with the involvement of a non-MMP proteinase, such as aggrecanase. The data suggest that in vitro MMPs may be involved in the base-line catabolism of aggrecan. Immunolocalization experiments showed that DIPEN341 and ITEGE373 epitopes were increased by treatment with IL-1α and retinoate. Confocal microscopy revealed that ITEGE373 epitope was largely intracellular but with matrix staining in the superficial zone, whereas DIPEN341epitope was cell-associated and widely distributed in the matrix. Surprisingly, the majority of 342FFGVG epitope, determined by radioimmunoassay and Western blotting, was retained in the tissue despite the absence of a G1 domain anchor. Interleukin-1α stimulation caused a marked increase in tissue DIPEN341 and342FFGVG epitope, and the 342FFGVG fragments retained in the tissue were larger than those released into the medium. Active porcine aggrecanase was unable to cleave342FFGVG fragments at the ↓Glu373↓Ala374 bond but cleaved intact aggrecan at this site, suggesting that 342FFGVG fragments are not substrates for aggrecanase. The apparent retention of large 342FFGVG fragments within cartilage, and their resistance to N-terminal cleavage by aggrecanase suggests that 342FF6V6 fragments may have a role in cartilage homeostasis.

Evidence of a novel aggrecan-degrading activity in cartilage: Studies of mice deficient in both ADAMTS-4 and ADAMTS-5

OBJECTIVE To characterize aggrecan catabolism and the overall phenotype in mice deficient in both ADAMTS-4 and ADAMTS-5 (TS-4/TS-5 Delta-cat) activity. METHODS Femoral head cartilage from the joints of TS-4/TS-5 Delta-cat mice and wild-type mice were cultured in vitro, and aggrecan catabolism was stimulated with either interleukin-1alpha (IL-1alpha) or retinoic acid. Total aggrecan release was measured, and aggrecanase activity was examined by Western blotting using neoepitope antibodies for detecting cleavage at EGE 373-374 ALG, SELE 1279-1280 GRG, FREEE 1467-1468 GLG, and AQE 1572-1573 AGEG. Aggrecan catabolism in vivo was examined by Western blotting of cartilage that had been extracted immediately ex vivo. RESULTS TS-4/TS-5 Delta-cat mice were viable, fertile, and phenotypically normal. TS-4/TS-5 Delta-cat cartilage explants did not release aggrecan in response to IL-1alpha, and there was no detectable increase in aggrecanase neoepitopes. TS-4/TS-5 Delta-cat cartilage explants released aggrecan in response to retinoic acid. There was no retinoic acid-stimulated cleavage at either EGE 373-374 ALG or AQE 1572-1573 AGEG. There was a low level of cleavage at SELE 1279-1280 GRG and major cleavage at FREEE 1467-1468 GLG. Ex vivo, cleavage at FREEE 1467-1468 GLG was substantially reduced, but still present, in TS-4/TS-5 Delta-cat mouse cartilage compared with wild-type mouse cartilage. CONCLUSION An aggrecanase other than ADAMTS-4 and ADAMTS-5 is expressed in mouse cartilage and is up-regulated by retinoic acid but not IL-1alpha. The novel aggrecanase appears to have different substrate specificity from either ADAMTS-4 or ADAMTS-5, cleaving E-G bonds but not E-A bonds. Neither ADAMTS-4 nor ADAMTS-5 is required for normal skeletal development or aggrecan turnover in cartilage.

Investigating ADAMTS-mediated aggrecanolysis in mouse cartilage

Proteolysis of the cartilage proteoglycan aggrecan is a feature of arthritis. We present a method for analyzing aggrecanolysis in in vitro cultures of 3-week-old mouse femoral head cartilage based on traditional methods developed for large animal species. Investigators can choose either a simple analysis that detects several aggrecan fragments released into culture medium only or a more comprehensive study that detects all fragments present in both the medium and the cartilage matrix. The protocol comprises (i) cartilage culture and optional cartilage extraction, (ii) a quick and simple colorimetric assay for quantitating aggrecan and (iii) neoepitope western blotting to identify specific aggrecan fragments partitioning to the medium or cartilage compartments. The crucial difference between the methods for mice and larger animals is that the proportion of aggrecan in a given sample is normalized to total aggrecan rather than to tissue wet weight. This necessary break from tradition arises because tiny volumes of liquid clinging to mouse cartilage can increase the apparent tissue wet weight, causing unacceptable errors. The protocol has broad application for the in vitro analysis of transgenic mice, particularly those with mutations that affect cartilage remodeling, arthritic disease and skeletal development. The protocol is robust, reliable and takes 7–11 d to complete.

Cortisol enhances structural maturation of the hypoplastic fetal lung in sheep

Although exogenous corticosteroids advance structural maturation of the fetal lung, they can adversely affect fetal lung and body growth. Our aim was to determine whether cortisol, at physiological doses, can enhance structural maturation of the hypoplastic fetal lung without affecting fetal lung growth. Fetal sheep were divided into four groups (n= 5 for each) and lung hypoplasia (LH) was induced in two groups. Increasing doses of cortisol (1.5–4.0 mg) were infused into one group of fetuses with LH and one group without LH; the other two groups received saline. LH retarded structural development, reduced tropoelastin mRNA levels, reduced hydroxyproline and elastin contents, and increased active matrix metalloproteinase‐2 (MMP‐2) levels in the fetal lung. Cortisol infusions had no effect on fetal lung growth or body weights. In fetuses with LH, cortisol increased the percentage airspace, reduced the interalveolar wall thickness, increased alveolar number and reduced the increase in active MMP‐2 levels. Thus, relatively low doses of cortisol can enhance structural maturation of the fetal lung without adversely affecting fetal lung growth. However, cortisol did not correct the abnormal deposition of elastin within the alveolar parenchyma associated with LH, indicating that secondary septal crest formation remained abnormal.

Neoepitope Antibodies Against MMP-Cleaved and Aggrecanase-Cleaved Aggrecan

Neoepitope antibodies recognize the newly created N or C terminus of protein degradation products but fail to recognize the same sequence of amino acids present in intact or undigested protein. Aggrecan neoepitope antibodies have been pivotal in studies determining the contribution of matrix metalloproteinases (MMPs) and aggrecanases to aggrecanolysis. In particular, an antibody to the A(374)RGSV N terminus was instrumental in the landmark discovery of the aggrecanases, ADAMTS-4 and ADAMTS-5. Antibodies to neoepitopes at the major MMP cleavage site DIPEN(341)/(342)FFGVG helped to distinguish MMP-driven aggrecan loss from aggrecanase-driven aggrecan loss and identified a role for MMPs in late-stage disease. More recently, neoepitope antibodies that recognize cleavage sites in the chondroitin sulphate-rich region of aggrecan have been used to show that aggrecanase cleavage proceeds in a defined manner, beginning at the C terminus and proceeding to the signature cleavage at NITEGE(373)/(374)ARGSV in the interglobular domain. Work with the C-terminal neoepitope antibodies has underscored the need to use a suite of neoepitope antibodies to fully describe aggrecanolysis in vitro. In this chapter, we describe the production of two aggrecan neoepitope antibodies as examples: the monoclonal anti-FFGVG antibody (AF-28) and the polyclonal anti-DIPEN antisera.

Matrix metalloproteinases are active following guanidine hydrochloride extraction of cartilage: generation of DIPEN neoepitope during dialysis.

We have recently observed marked increases in MMP-derived aggrecan fragments in extracts of cartilage stimulated with IL-1. The fragments were detected with an anti-DIPEN neoepitope antibody that is specific for fragments generated by MMP cleavage at the DIPEN(341) F(342)FGVG site. Because our results contrasted with another study, we systematically compared our methods with other published methods. We now report that DIPEN(341) neoepitope can be generated post-culture, by dialysing GuHCl(1)-denatured samples against unbuffered, deionized water at 4 degrees C. We show that EDTA must be included in the GuHCl extractant, as well as the dialysis buffer, in order to block post-culture processing of aggrecan by MMPs.

A Disintegrin and Metalloproteinase with Thrombospondin Motifs-5 (ADAMTS-5) Forms Catalytically Active Oligomers

The metalloproteinase ADAMTS-5 (A disintegrin and metalloproteinase with thrombospondin motifs) degrades aggrecan, a proteoglycan essential for cartilage structure and function. ADAMTS-5 is the major aggrecanase in mouse cartilage, and is also likely to be the major aggrecanase in humans. ADAMTS-5 is a multidomain enzyme, but the function of the C-terminal ancillary domains is poorly understood. We show that mutant ADAMTS-5 lacking the catalytic domain, but with a full suite of ancillary domains inhibits wild type ADAMTS activity, in vitro and in vivo, in a dominant-negative manner. The data suggest that mutant ADAMTS-5 binds to wild type ADAMTS-5; thus we tested the hypothesis that ADAMTS-5 associates to form oligomers. Co-elution, competition, and in situ PLA experiments using full-length and truncated recombinant ADAMTS-5 confirmed that ADAMTS-5 molecules interact, and showed that the catalytic and disintegrin-like domains support these intermolecular interactions. Cross-linking experiments revealed that recombinant ADAMTS-5 formed large, reduction-sensitive oligomers with a nominal molecular mass of ∼400 kDa. The oligomers were unimolecular and proteolytically active. ADAMTS-5 truncates comprising the disintegrin and/or catalytic domains were able to competitively block full-length ADAMTS-5-mediated aggrecan cleavage, measured by production of the G1-EGE373 neoepitope. These results show that ADAMTS-5 oligomerization is required for full aggrecanase activity, and they provide evidence that blocking oligomerization inhibits ADAMTS-5 activity. The data identify the surface provided by the catalytic and disintegrin-like domains of ADAMTS-5 as a legitimate target for the design of aggrecanase inhibitors.

Proteoglycan and Collagen Degradation in Osteoarthritis

The gradual loss of articular cartilage from the surface of articulating joints is a feature of osteoarthritis. It is marked by degradation of the cartilage matrix, including the large aggregating proteoglycan aggrecan, the small leucine-rich proteoglycans known as SLRPs and the fibrillar type II collagen. Aggrecan provides the water-holding capacity of cartilage, while the collagen II scaffold provides elastic restraint, aided by a protective coat of small leucine-rich proteoglycans. Damaged aggrecan is readily replaced by synthesis of new aggrecan; however, type II collagen can resist only a limited amount of proteolysis before cartilage function is compromised. In this review the major enzyme families of cartilage-degrading enzymes, the matrix metalloproteinases (MMPs) and a disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS) families, are discussed. We examine factors that regulate MMP and ADAMTS activity, with a focus on MMP-13, ADAMTS-4 and ADAMTS-5 as the major protagonists of cartilage degradation. We also compare the effects of blocking aggrecanolysis and collagenolysis separately, or together, on cartilage erosion in a mouse model of osteoarthritis. The role of degraded matrix fragments in regulating inflammation in osteoarthritis, via Toll-like receptor signalling, is also discussed.