Suzanne B. Golub

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.

Blocking aggrecanase cleavage in the aggrecan interglobular domain abrogates cartilage erosion and promotes cartilage repair

Aggrecan loss from cartilage in arthritis is mediated by aggrecanases. Aggrecanases cleave aggrecan preferentially in the chondroitin sulfate-2 (CS-2) domain and secondarily at the E(373) downward arrow(374)A bond in the interglobular domain (IGD). However, IGD cleavage may be more deleterious for cartilage biomechanics because it releases the entire CS-containing portion of aggrecan. Recent studies identifying aggrecanase-2 (ADAMTS-5) as the predominant aggrecanase in mouse cartilage have not distinguished aggrecanolysis in the IGD from aggrecanolysis in the CS-2 domain. We generated aggrecan knockin mice with a mutation that rendered only the IGD resistant to aggrecanases in order to assess the contribution of this specific cleavage to cartilage pathology. The knockin mice were viable and fertile. Aggrecanase cleavage in the aggrecan IGD was not detected in knockin mouse cartilage in situ nor following digestion with ADAMTS-5 or treatment of cartilage explant cultures with IL-1 alpha. Blocking cleavage in the IGD not only diminished aggrecan loss and cartilage erosion in surgically induced osteoarthritis and a model of inflammatory arthritis, but appeared to stimulate cartilage repair following acute inflammation. We conclude that blocking aggrecanolysis in the aggrecan IGD alone protects against cartilage erosion and may potentiate cartilage repair.

Hyaluronan synthesis and degradation in cartilage and bone

Abstract.Hyaluronan (HA) is a large but simple glycosaminoglycan composed of repeating D-glucuronic acid, β1–3 linked to N-acetyl-D-glucosamine β1–4, found in body fluids and tissues, in both intra- and extracellular compartments. Despite its structural simplicity, HA has diverse functions in skeletal biology. In development, HA-rich matrices facilitate migration and condensation of mesenchymal cells, and HA participates in joint cavity formation and longitudinal bone growth. In adult cartilage, HA binding to aggrecan immobilises aggrecan, retaining it at the high concentrations required for compressive resilience. HA also appears to regulate bone remodelling by controlling osteoclast, osteoblast and osteocyte behaviour. The functions of HA depend on its intrinsic properties, which in turn rely on the degree of polymerisation by HA synthases, depolymerisation by hyaluronidases, and interactions with HA-binding proteins. HA synthesis and degradation are closely regulated in skeletal tissues and aberrant synthetic or degradative activity causes disease. The role and regulation of HA synthesis and degradation in cartilage, bone and skeletal development is discussed.

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.

ADAMTS-5 deficiency does not block aggrecanolysis at preferred cleavage sites in the chondroitin sulfate-rich region of aggrecan.

In the mouse, proteolysis in the aggrecan interglobular domain is driven by ADAMTS-5, and mice deficient in ADAMTS-5 catalytic activity are protected against aggrecan loss and cartilage damage in experimental models of arthritis. Here we show that despite ablation of ADAMTS-5 activity, aggrecanolysis can still occur at two preferred sites in the chondroitin sulfate-rich region. Retinoic acid was more effective than interleukin-1α (IL) in promoting cleavage at these sites in ADAMTS-5-deficient cartilage. These results suggest that cleavage at preferred sites in the chondroitin sulfate-rich region is mediated by ADAMTS-4 or an aggrecanase other than ADAMTS-5. Following retinoic acid or IL-1α stimulation of cartilage explants, aggrecan fragments in medium and extracts contained SELE1279 or FREEE1467 C-terminal sequences. Some SELE1279 and FREEE1467 fragments were retained in the cartilage, with intact G1 domains. Other SELE1279 fragments were released into the medium and co-migrated with the 374ALGS neoepitope, indicating they were aggrecanase-derived fragments. In contrast none of the FREEE1467 fragments released into the medium co-migrated with the 374ALGS neoepitope, suggesting that, despite their size, these fragments were not products of aggrecanase cleavage in the interglobular domain. ADAMTS-5, but not ADAMTS-1, -4, or -9, was up-regulated 8-fold by retinoic acid and 17-fold by IL-1α treatment. The data show that whereas ADAMTS-5 is entirely responsible for cleavage in the interglobular domain, cleavage in the chondroitin sulfate-rich region is driven either by ADAMTS-4, which compensates for loss of ADAMTS-5 in this experimental system, or possibly by another aggrecanase. The data show that there are differential aggrecanase activities with preferences for separate regions of the core protein.

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.

Deposition and selective degradation of structually-abnormal type I collagen in a collagen matrix produced by osteogenesis imperfecta fibroblasts in vitro

Collagen matrix deposition and turnover were studied in skin fibroblasts from a control and from a patient with lethal perinatal osteogenesis imperfecta (OI) identified as a Gly667 to Arg substitution in the alpha 1(I) chain. A culture system where ascorbic acid was included to stimulate collagen matrix formation over extended culture periods was used. Serial extraction of the control cell collagen matrix confirmed that a substantial mature crosslinked collagen matrix was formed in the control fibroblast cell layer. In contrast, total collagen deposition by the OI fibroblasts was poor, with the quantity of collagen deposited only about a quarter of that of the control cells. Detailed analysis of the OI fibroblast matrix revealed that the mutant collagen chains were incorporated into the collagenous matrix. These data indicate that, when grown with ascorbate in long-term culture, OI fibroblasts reproduced the abnormal matrix deposition pattern of OI tissues in vivo. The overall dramatic reduction in collagen matrix formation was not accounted for by reduced collagen production, since during the period of matrix deposition (days 8-12) the rate of production by the OI cells was only slightly less than that of the control cells. The incorporation of the newly-synthesized OI collagen into the matrix was less efficient than in control cells, reflecting the cooperative nature of matrix deposition. The fate of this mutant collagen containing the Gly to Arg charge-change was followed in the matrix by a pulse-chase experiment and two-dimensional electrophoresis. These data demonstrated that the mutant incorporated into the matrix was unstable, with the proportion of mutant declining during the chase. The deposition of the mutant monomers into a pool more accessible to proteolytic degradation indicated that the mutant and normal collagens did not copolymerize to form collagen fibers of even collagen distribution, but rather the mutant collagen was either enriched on the exposed surfaces of mixed-composition fibers, or was unable to form copolymers efficiently and polymerized into mutant-only fibrillar assemblies more prone to proteolytic attack.

Bioactivity in an Aggrecan 32-mer Fragment Is Mediated via Toll-like Receptor 2.

To determine whether an aggrecan 32‐mer fragment derived from dual ADAMTS and matrix metalloproteinase (MMP) cleavage in the aggrecan interglobular domain was bioactive and, if so, to elucidate its mechanism of action.

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.

Abundant LacZ activity in the absence of Cre expression in the normal and inflamed synovium of adult Col2a1-Cre; ROSA26RLacZ reporter mice

Recent analyses of Col2a1-Cre; ROSA26R reporter mice showed that synovial fibroblasts in 7-day mice were LacZ positive, due to a history of Col2a1-Cre expression conferred by their origin in the interzone of the developing joint. We have examined LacZ staining in adult Col2a1-Cre(+/0); ROSA26R(LacZ) mice, with and without inflammatory arthritis, and found that synovial fibroblasts in normal and inflamed synovium are LacZ positive, but Cre negative. Our results suggest that Cre-mediated recombination in joint interzone cells during development endure in adult synovial cells despite the absence of ongoing Cre expression. These findings have important implications and applications for the study of synovial inflammation in models of experimental arthritis.