Aggrecanases and cartilage matrix degradation

The loss of extracellular matrix macromolecules from the cartilage results in serious impairment of joint function. Metalloproteinases called 'aggrecanases' that cleave the Glu³⁷³–Ala³⁷⁴ bond of the aggrecan core protein play a key role in the early stages of cartilage destruction in rheumatoid arthritis and in osteoarthritis. Three members of the ADAMTS family of proteinases, ADAMTS-1, ADAMTS-4 and ADAMTS-5, have been identified as aggrecanases. Matrix metalloproteinases, which are also found in arthritic joints, cleave aggrecans, but at a distinct site from the aggrecanases (i.e. Asn³⁴¹–Phe³⁴²). The present review discuss the enzymatic properties of the three known aggrecanases, the regulation of their activities, and their role in cartilage matrix breakdown during the development of arthritis in relation to the action of matrix metalloproteinases.     

ADAMTS-12 associates with and degrades cartilage oligomeric matrix protein

Loss of articular cartilage because of extracellular matrix breakdown is the hallmark of arthritis. Degradative fragments of cartilage oligomeric matrix protein (COMP), a prominent noncollagenous matrix component in articular cartilage, have been observed in the cartilage, synovial fluid, and serum of arthritis patients. The molecular mechanism of COMP degradation and the enzyme(s) responsible for it, however, remain largely unknown. ADAMTS-12 (a disintegrin and metalloprotease with thrombospondin motifs) was shown to associate with COMP both in vitro and in vivo. ADAMTS-12 selectively binds to only the epidermal growth factor-like repeat domain of COMP of the four functional domains tested. The four C-terminal TSP-1-like repeats of ADAMTS-12 are shown to be necessary and sufficient for its interaction with COMP. Recombinant ADAMTS-12 is capable of digesting COMP in vitro. The COMP-degrading activity of ADAMTS-12 requires the presence of Zn2+ and appropriate pH (7.5-9.5), and the level of ADAMTS-12 in the cartilage and synovium of patients with both osteoarthritis and rheumatoid arthritis is significantly higher than in normal cartilage and synovium. Together, these findings indicate that ADAMTS-12 is a new COMP-interacting and -degrading enzyme and thus may play an important role in the COMP degradation in the initiation and progression of arthritis.     

Comparison of age-dependent expression of aggrecan and ADAMTSs in mandibular condylar cartilage, tibial growth plate, and articular cartilage in rats

A disintegrin and metalloproteinase with thrombospondin motif (adamalysin–thrombospondins, ADAMTS) degrades aggrecan, one of the major extracellular matrix (ECM) components in cartilage. Mandibular condylar cartilage differs from primary cartilage, such as articular and growth plate cartilage, in its metabolism of ECM, proliferation, and differentiation. Mandibular condylar cartilage acts as both articular and growth plate cartilage in the growing period, while it remains as articular cartilage after growth. We hypothesized that functional and ECM differences between condylar and primary cartilages give rise to differences in gene expression patterns and levels of aggrecan and ADAMTS-1, -4, and -5 during growth and aging. We employed in situ hybridization and semiquantitative RT-PCR to identify mRNA expression for these molecules in condylar cartilage and primary cartilages during growth and aging. All of the ADAMTSs presented characteristic, age-dependent expression patterns and levels among the cartilages tested in this study. ADAMTS-5 mainly contributed to ECM metabolism in growth plate and condylar cartilage during growth. ADAMTS-1 and ADAMTS-4 may be involved in ECM turn over in articular cartilage. The results of the present study reveal that ECM metabolism and expression of related proteolytic enzymes in primary and secondary cartilages may be differentially regulated during growth and aging.     

Interleukin-4 antagonizes oncostatin M and transforming growth factor beta-induced responses in articular chondrocytes

Oncostatin M (OSM) stimulates cartilage degradation in rheumatoid arthritis (RA) by inducing matrix metalloproteinases (MMPs) and aggrecanases (ADAMTS; a disintegrin and metalloproteinase with thrombospondin motif). Transforming growth factor beta (TGF-beta1) induces cartilage repair in joints but in excessive amounts, promotes inflammation. OSM and TGF-beta1 also induce tissue inhibitor of metalloproteinase-3 (TIMP-3), an important natural inhibitor of MMPs, aggrecanases, and tumor necrosis factor alpha converting enzyme (TACE), the principal proteases involved in arthritic inflammation and cartilage degradation. We studied cartilage protective mechanisms of the antiinflammatory cytokine, interleukin-4 (IL-4). IL-4 strongly (MMP-13 and TIMP-3) or minimally (ADAMTS-4) suppressed OSM-induced gene expression in chondrocytes. IL-4 did not affect OSM-stimulated phosphorylation of extracellular signal-regulated kinases (ERKs), protein 38 (p38), c-Jun N-terminal kinase (JNK) and Stat1. Lack of additional suppression with their inhibitors suggested that MMP-13, ADAMTS-4, and TIMP-3 inhibition was independent of these mediators. IL-4 also downregulated TGF-beta1-induced TIMP-3 gene expression, Smad2, and JNK phosphorylation. Additional suppression of TIMP-3 RNA by JNK inhibitor suggests JNK implication. The cartilage protective effects of IL-4 in animal models of arthritis may be due to its inhibition of MMPs and ADAMTS-4 expression. However, suppression of TIMP-3 suggests caution for using IL-4 as a cartilage protective therapy.     

MMP-13 is constitutively produced in human chondrocytes and co-endocytosed with ADAMTS-5 and TIMP-3 by the endocytic receptor LRP1

Matrix metalloproteinase 13 (MMP-13) degrades collagenous extracellular matrix and its aberrant activity associates with diseases such as arthritis, cancer, atherosclerosis and fibrosis. The wide range of MMP-13 proteolytic capacity suggests that it is a powerful, potentially destructive proteinase and thus it has been believed that MMP-13 is not produced in most adult human tissues in the steady state. Present study has revealed that human chondrocytes isolated from healthy adults constitutively express and secrete MMP-13, but that it is rapidly endocytosed and degraded by chondrocytes. Both pro- and activated MMP-13 bind to clusters II and III of low-density lipoprotein (LDL) receptor-related protein 1 (LRP1). Domain deletion studies indicated that the hemopexin domain is responsible for this interaction. Binding competition between MMP-13 and ADAMTS-4, -5 or TIMP-3, which also bind to cluster II, further shown that the MMP-13 binding site within cluster II is different from those of ADAMTS-4, -5 or TIMP-3. MMP-13 is therefore co-endocytosed with ADAMTS-5 and TIMP-3 by human chondrocytes. These findings indicate that MMP-13 may play a role on physiological turnover of cartilage extracellular matrix and that LRP1 is a key modulator of extracellular levels of MMP-13 and its internalization is independent of the levels of ADAMTS-4, -5 and TIMP-3.     

ADAMTS Expression in Colorectal Cancer

ADAMTSs are a family of secreted proteinases that share the metalloproteinase domain with matrix metalloproteinases (MMPs). By acting on a large panel of extracellular substrates, they control several cell functions such as fusion, adhesion, proliferation and migration. Through their thrombospondin motifs they also possess anti-angiogenic properties. We investigated whether ADAMTSs participate in colorectal cancer progression and invasion. Their expression was investigated at both mRNA and protein levels. Using RT-PCR, the expression of ADAMTS-1, -4, -5 and ADAMTS-20 was estimated in colorectal tumors of different cancer stage and anatomic site and 3 cell lines of different aggressiveness. An overexpression of ADAMTS-4 and -5 was observed, especially in tissue samples, whereas ADAMTS-1 and -20 were found to be down-regulated. Western blot analysis further supported the RT-PCR findings, revealing in addition the degradation of ADAMTS-1 and -20 in cancer. In situ expression and localization of ADAMTS-1, -4, -5 and -20 was also investigated by immunohistochemical analysis. Our data suggest a positive correlation between ADAMTS-4 and -5 expression and cancer progression, in contrast with the anti-angiogenic members of the family, ADAMTS-1 and -20, which were found to be down-regulated. Our findings support the notion that overexpression of ADAMTS-4 and ADAMTS-5 in colorectal cancer might be a possible invasive mechanism of cancer cells in order to degrade proteoglycans of ECM.     

Proteolytic activities of human ADAMTS-5: comparative studies with ADAMTS-4

Aggrecanases have been characterized as proteinases that cleave the Glu373-Ala374 bond of the aggrecan core protein, and they are multidomain metalloproteinases belonging to the ADAMTS (adamalysin with thrombospondin type 1 motifs) family. The first aggrecanases discovered were ADAMTS-4 (aggrecanase 1) and ADAMTS-5 (aggrecanase 2). They contain a zinc catalytic domain followed by non-catalytic ancillary domains, including a disintegrin domain, a thrombospondin domain, a cysteine-rich domain, and a spacer domain. In the case of ADAMTS-5, a second thrombospondin domain follows the spacer domain. We previously reported that the non-catalytic domains of ADAMTS-4 influence both its extracellular matrix interaction and proteolytic abilities. Here we report the effects of these domains of ADAMTS-5 on the extracellular matrix interaction and proteolytic activities and compare them with those of ADAMTS-4. Although the spacer domain was critical for ADAMTS-4 localization in the matrix, the cysteine-rich domain influenced ADAMTS-5 localization. Similar to previous reports of other ADAMTS family members, very little proteolytic activity was detected with the ADAMTS-5 catalytic domain alone. The sequential inclusion of each carboxyl-terminal domain enhanced its activity against aggrecan, carboxymethylated transferrin, fibromodulin, decorin, biglycan, and fibronectin. Both ADAMTS-4 and -5 had a broad optimal activity at pH 7.0-9.5. Aggrecanolytic activities were sensitive to the NaCl concentration, but activities on non-aggrecan substrates, e.g. carboxymethylated transferrin, were not affected. Although ADAMTS-4 and ADAMTS-5 had similar general proteolytic activities, the aggrecanase activity of ADAMTS-5 was at least 1,000-fold greater than that of ADAMTS-4 under physiological conditions. Our studies suggest that ADAMTS-5 is a major aggrecanase in cartilage metabolism and pathology.     

Leptin Downregulates Aggrecan through the p38-ADAMST Pathway in Human Nucleus Pulposus Cells

The mechanistic basis of obesity-associated intervertebral disc degeneration (IDD) is unclear. Aberrant expression of aggrecan and its degrading enzymes ADAMTS-4 and ADAMTS-5 is implicated in the development of IDD. Here, we investigated the effect of leptin, a hormone with increased circulating levels in obesity, on the expression of aggrecan and ADAMTSs in primary human nucleus pulposus (NP) cells. Real-time PCR and Western blots showed that leptin increased the mRNA and protein expression of ADAMTS-4 and ADAMTS-5 and reduced the level of aggrecan in NP cells, accompanied by a prominent induction of p38 phosphorylation. Treatment of NP cells with SB203580 (a p38 inhibitor) abolished the regulation of aggrecan and ADAMTSs by leptin. Knockdown of ADAMTS-4 and ADAMTS-5 by siRNAs also attenuated the degradation of aggrecan in leptin-stimulated NP cells. To conclude, we demonstrated that leptin induces p38 to upregulate ADAMTSs and thereby promoting aggrecan degradation in human NP cells. These results provide a novel mechanistic insight into the molecular pathogenesis of obesity-associated IDD.     

Matrilin-4 is processed by ADAMTS-5 in late Golgi vesicles present in growth plate chondrocytes of defined differentiation state

The two aggrecanases ADAMTS-4 and ADAMTS-5 have been shown to not only play roles in the breakdown of cartilage extracellular matrix in osteoarthritis, but also mediate processing of matrilins in the secretory pathway. The matrilins are adaptor proteins with a function in connecting fibrillar and network-like components in the cartilage extracellular matrix. Cleavage resulting in processed matrilins with fewer ligand-binding subunits could make these less efficient in providing matrix cohesion. In this study, the processing and degradation of matrilin-4 during cartilage remodeling in the growth plate of the developing mouse long bones were studied in greater detail. We show that ADAMTS-5 and a matrilin-4 neoepitope, revealed upon ADAMTS cleavage, colocalize in prehypertrophic/hypertrophic chondrocytes while they are not detected in proliferating chondrocytes of the growth plate. ADAMTS-5 and the cleaved matrilin-4 are preferentially detected in vesicles derived from the Golgi apparatus. The matrilin-4 neoepitope was not observed in the growth plate of ADAMTS-5 deficient mice. We propose that in the growth plate ADAMTS-5, and not ADAMTS-4, has a physiological function in the intracellular processing of matrilins and potentially of other extracellular matrix proteins.     

ADAMTS proteinases: a multi-domain,multi-functional family with roles in extracellular matrix turnover and arthritis

Members of the ADAMTS (a disintegrin and metalloproteinase with thrombospondin motifs) family are known to influence development, angiogenesis, coagulation and progression of arthritis. As proteinases their substrates include the von Willebrand factor precursor and extracellular matrix components such as procollagen, hyalectans (hyaluronan-binding proteoglycans including aggrecan), decorin, fibromodulin and cartilage oligomeric matrix protein. ADAMTS levels and activities are regulated at multiple levels through the control of gene expression, mRNA splicing, protein processing and inhibition by TIMP (tissue inhibitor of metalloproteinases). A recent screen of human cartilage has shown that multiple members of the ADAMTS family may be important in connective tissue homeostasis and pathology.     

ADAMTS-4 and ADAMTS-5: key enzymes in osteoarthritis

Osteoarthritis (OA) is a progressive disease of the joints characterized by degradation of articular cartilage. Although disease initiation may be multi-factorial, the cartilage destruction appears to be a result of uncontrolled proteolytic extracellular matrix destruction. A major component of the cartilage extracellular matrix is aggrecan, a proteoglycan that imparts compressive resistance to the tissue. Aggrecanase-mediated aggrecan degradation is a significant event in early stage OA. The relative contribution of individual ADAMTS-4 and ADAMTS-5 proteinases to cartilage destruction during OA has not been resolved completely. This review reveals that both ADAMTS-4/ADAMTS-5 are responsible for aggrecan degradation in a human model of OA, and is expected to list down the rational strategies which are being focussed for therapeutic intervention in OA.     

Screening of potential a disintegrin and metalloproteinase with thrombospondin motifs-4 inhibitors using a collagen model fluorescence resonance energy transfer substrate

The major components of the cartilage extracellular matrix are type II collagen and aggrecan. Type II collagen provides cartilage with its tensile strength, whereas the water-binding capacity of aggrecan provides compressibility and elasticity. Aggrecan breakdown leads to an increase in proteolytic susceptibility of articular collagen; hence, aggrecan may also have a protective effect on type II collagen. Given their role in aggrecan degradation and differing substrate specificity profiles, the pursuit of inhibitors for both aggrecanase 1 (a disintegrin and metalloproteinase with thrombospondin motifs-4 [ADAMTS-4]) and aggrecanase 2 (ADAMTS-5) is desirable. We previously described collagen model fluorescence resonance energy transfer (FRET) substrates for aggrecan-degrading members of the ADAMTS family. These FRET substrate assays are also fully compatible with multiwell formats. In the current study, a collagen model FRET substrate was examined for inhibitor screening of ADAMTS-4. ADAMTS-4 was screened against a small compound library (n=960) with known pharmacological activity. Five compounds that inhibited ADAMTS-4>60% at a concentration of 1muM were identified. A secondary screen using reversed-phase high-performance liquid chromatography (RP-HPLC) was developed and performed for verification of the five potential inhibitors. Ultimately, piceatannol was confirmed as a novel inhibitor of ADAMTS-4, with an IC(50) value of 1muM. Because the collagen model FRET substrates have distinct conformational features that may interact with protease secondary substrate sites (exosites), nonactive site-binding inhibitors can be identified via this approach. Selective inhibitors for ADAMTS-4 would allow a more definitive evaluation of this protease in osteoarthritis and also represent a potential next generation in metalloproteinase therapeutics.     

Cleavage of cartilage oligomeric matrix protein (thrombospondin-5) by matrix metalloproteinases and a disintegrin and metalloproteinase with thrombospondin motifs.

Cartilage oligomeric matrix protein (COMP) is a pentameric glycoprotein present in cartilage, tendon and ligament. Fragments of the molecule are present in the diseased cartilage, synovial fluid and serum of patients with knee injuries, osteoarthritis and rheumatoid arthritis. Although COMP is a substrate for several matrix metalloproteinases (MMPs), the enzymes responsible for COMP degradation in vivo have yet to be identified. In this study we utilised well-established bovine cartilage culture models to examine IL-1alpha-stimulated COMP proteolysis in the presence and absence of MMP inhibitors. COMP was released from bovine nasal cartilage, in response to IL-1alpha, at an intermediate time between proteoglycans and type II collagen, when soluble MMP levels in the culture medium were undetectable. The major fragment of COMP released following IL-1alpha-stimulation migrated with an apparent molecular mass of approximately 110 kDa (Fragment-110) and co-migrated with both the major fragment present in human arthritic synovial fluid samples and the product of COMP cleavage by purified MMP-9. However, the broad-spectrum MMP and ADAM inhibitor BB94 only partially inhibited the formation of Fragment-110 and failed to inhibit COMP release significantly. Therefore the results of these studies indicate a role for proteinases other than MMPs in the degradation of COMP in bovine cartilage. It was further demonstrated that purified COMP was cleaved by ADAMTS-4, but not ADAMTS-1 or -5, to yield a fragment which co-migrated with Fragment-110. Therefore this is the first demonstration of COMP as a substrate for ADAMTS-4, although it remains to be determined whether this enzyme plays a role in COMP degradation in vivo.     

Alpha2-macroglobulin is a novel substrate for ADAMTS-4 and ADAMTS-5 and represents an endogenous inhibitor of these enzymes

Osteoarthritis is characterized by the loss of aggrecan and collagen from the cartilage extracellular matrix. The proteinases responsible for the breakdown of cartilage aggrecan include ADAMTS-4 (aggrecanase 1) and ADAMTS-5 (aggrecanase 2). Post-translational inhibition of ADAMTS-4/-5 activity may be important for maintaining normal homeostasis of aggrecan metabolism, and thus, any disruption to this inhibition could lead to accelerated aggrecan breakdown. To date TIMP-3 (tissue inhibitor of matrix metalloproteinases-3) is the only endogenous inhibitor of ADAMTS-4/-5 that has been identified. In the present studies we identify alpha(2)-macroglobulin (alpha(2)M) as an additional endogenous inhibitor of ADAMTS-4 and ADAMTS-5. alpha(2)M inhibited the activity of both ADAMTS-4 and ADAMTS-5 in a concentration-dependent manner, demonstrating 1:1 stoichiometry with second-order rate constants on the order of 10(6) and 10(5) m(-1) s(-1), respectively. Inhibition of the aggrecanases was mediated by proteolysis of the bait region within alpha(2)M, resulting in physical entrapment of these proteinases. Both ADAMTS-4 and ADAMTS-5 cleaved alpha(2)M at Met(690)/Gly(691), representing a novel proteinase cleavage site within alpha(2)M and a novel site of cleavage for ADAMTS-4 and ADAMTS-5. Finally, the use of the anti-neoepitope antibodies to detect aggrecanase-generated alpha(2)M-fragments in synovial fluid was investigated and found to be uninformative.     

Syndecan-4 in intervertebral disc and cartilage: Saint or synner?

The ECM of the intervertebral disc and articular cartilage contains a highly organised network of collagens and proteoglycans which resist compressive forces applied to these tissues. A pathological hallmark of the intervertebral disc is the imbalance between production of anabolic and catabolic factors by the resident cells. This process is thought to be mediated by pro-inflammatory cytokines, predominantly TNF-α and IL-1β, which upregulate expression of matrix degrading enzymes such as MMPs and ADAMTSs. This imbalance ultimately results in tissue degeneration causing failure of the biomechanical function of the tissues. A similar cascade of events is thought to occur in articular cartilage during development of osteoarthritis. Within these skeletal tissues a small, cell surface heparan sulphate proteoglycan; syndecan-4 (SDC4) has been implicated in maintaining physiological functions. However in the degenerating niche of the intervertebral disc and cartilage, dysregulated activities of this molecule may exacerbate pathological changes. Studies in recent years have elucidated a role for SDC4 in mediating matrix degradation in both intervertebral discs and cartilage by controlling ADAMTS-5 function and MMP3 expression. Discourse presented in this review highlights the potential of SDC4 as a possible therapeutic target in slowing the progression of ECM degradation in both degenerative disc disease and osteoarthritis.