Amanda J. Fosang

Proteoglycans: many forms and many functions.

Proteoglycans are produced by most eukaryotic cells and are versatile components of pericellular and extracellular matrices. They belong to many different protein families. Their functions vary from the physical effects of the proteoglycan aggrecan, which binds with link protein to hyaluronan to form multi‐molecular aggregates in cartilage; to the intercalated membrane protein CD44 that has a proteoglycan form and is a receptor and a cell‐binding site for hyaluronan; to heparan sulfate proteoglycans of the syndecan and other families that provide matrix binding sites and cell‐surface receptors for growth factors such as fibroblast growth factor (FGF). One feature that recurs in proteoglycan biology is that their structure is open to extensive modulation during cellular expression. Examples of protein changes are known, but a major source of structural variation is in the glycosaminoglycan chains. The number of chains and their length can vary, as well as their pattern of sulfation. This may result in the switching of different chain types with different properties, e.g., chondroitin sulfate and heparan sulfate, and it may also result in the selective expression of sulfated chain sequences that have specific functions. The control of glycosaminoglycan structure is not well understood, but it does appear to be used to change the properties of proteoglycans to suit different biological needs. Proteoglycan forms of proteins are thus important modifiers of the organization of the pericellular and extracellular matrices and modulators of the processes that occur there.—Hardingham, T. E., Fosang, A. J. Protoglycans: many forms and many functions. FASEB J. 6: 861‐870; 1992.

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.

Altered endochondral bone development in matrix metalloproteinase 13-deficient mice

The assembly and degradation of extracellular matrix (ECM) molecules are crucial processes during bone development. In this study, we show that ECM remodeling is a critical rate-limiting step in endochondral bone formation. Matrix metalloproteinase (MMP) 13 (collagenase 3) is poised to play a crucial role in bone formation and remodeling because of its expression both in terminal hypertrophic chondrocytes in the growth plate and in osteoblasts. Moreover, a mutation in the human MMP13 gene causes the Missouri variant of spondyloepimetaphyseal dysplasia. Inactivation of Mmp13 in mice through homologous recombination led to abnormal skeletal growth plate development. Chondrocytes differentiated normally but their exit from the growth plate was delayed. The severity of the Mmp13- null growth plate phenotype increased until about 5 weeks and completely resolved by 12 weeks of age. Mmp13-null mice had increased trabecular bone, which persisted for months. Conditional inactivation of Mmp13 in chondrocytes and osteoblasts showed that increases in trabecular bone occur independently of the improper cartilage ECM degradation caused by Mmp13 deficiency in late hypertrophic chondrocytes. Our studies identified the two major components of the cartilage ECM, collagen type II and aggrecan, as in vivo substrates for MMP13. We found that degradation of cartilage collagen and aggrecan is a coordinated process in which MMP13 works synergistically with MMP9. Mice lacking both MMP13 and MMP9 had severely impaired endochondral bone, characterized by diminished ECM remodeling, prolonged chondrocyte survival, delayed vascular recruitment and defective trabecular bone formation (resulting in drastically shortened bones). These data support the hypothesis that proper ECM remodeling is the dominant rate-limiting process for programmed cell death, angiogenesis and osteoblast recruitment during normal skeletal morphogenesis.

Degradation of cartilage aggrecan by collagenase-3 (MMP-13)

Degradation of the large cartilage proteoglycan aggrecan in arthritis involves an unidentified enzyme aggrecanase, and at least one of the matrix metalloproteinases. Proteinase‐sensitive cleavage sites in the aggrecan interglobular domain (IGD) have been identified for many of the human MMPs, as well as for aggrecanase and other proteinases. The major MMP expressed by chondrocytes stimulated with retinoic acid to degrade their matrix is collagenase‐3 or MMP‐13. Because of its potential role in aggrecan degradation we examined the specificity of MMP‐13 for an aggrecan substrate. The results show that MMP‐13 cleaves aggrecan in the IGD at the same site (..PEN341‐FFG..) identified for other members of the MMP family, and also at a novel site ..VKP384‐VFE.. not previously observed for other proteinases.

Matrix metalloproteinase 13–deficient mice are resistant to osteoarthritic cartilage erosion but not chondrocyte hypertrophy or osteophyte development

OBJECTIVE To investigate the role of matrix metalloproteinase 13 (MMP-13; collagenase 3) in osteoarthritis (OA). METHODS OA was surgically induced in the knees of MMP-13-knockout mice and wild-type mice, and mice were compared. Histologic scoring of femoral and tibial cartilage aggrecan loss (0-3 scale), erosion (0-7 scale), and chondrocyte hypertrophy (0-1 scale), as well as osteophyte size (0-3 scale) and maturity (0-3 scale) was performed. Serial sections were stained for type X collagen and the MMP-generated aggrecan neoepitope DIPEN. RESULTS Following surgery, aggrecan loss and cartilage erosion were more severe in the tibia than femur (P<0.01) and tibial cartilage erosion increased with time (P<0.05) in wild-type mice. Cartilaginous osteophytes were present at 4 weeks and underwent ossification, with size and maturity increasing by 8 weeks (P<0.01). There was no difference between genotypes in aggrecan loss or cartilage erosion at 4 weeks. There was less tibial cartilage erosion in knockout mice than in wild-type mice at 8 weeks (P<0.02). Cartilaginous osteophytes were larger in knockout mice at 4 weeks (P<0.01), but by 8 weeks osteophyte maturity and size were no different from those in wild-type mice. Articular chondrocyte hypertrophy with positive type X collagen and DIPEN staining occurred in both wild-type and knockout mouse joints. CONCLUSION Our findings indicate that structural cartilage damage in a mouse model of OA is dependent on MMP-13 activity. Chondrocyte hypertrophy is not regulated by MMP-13 activity in this model and does not in itself lead to cartilage erosion. MMP-13 deficiency can inhibit cartilage erosion in the presence of aggrecan depletion, supporting the potential for therapeutic intervention in established OA with MMP-13 inhibitors.

Aggrecan is degraded by matrix metalloproteinases in human arthritis. Evidence that matrix metalloproteinase and aggrecanase activities can be independent.

Proteolytic degradation of aggrecan is a hallmark of the pathology of arthritis, yet the identity of the enzyme(s) in cartilage responsible for this degradation is unknown. Previous studies have suggested that the matrix metalloproteinases (MMPs) may be involved but there has been no definitive evidence for their direct action in the proteolysis of aggrecan in human arthritis. We now show unequivocally that aggrecan fragments derived from the specific action of MMPs can be detected in synovial fluids from patients with both inflammatory and noninflammatory arthritis, with a neoepitope monoclonal antibody AF-28 that detects the NH2-terminal sequence F342FGVG.... The synovial fluid MMP fragments were of low buoyant density and distributed exclusively at the top of cesium chloride density gradients, suggesting that these fragments lacked chondroitin sulfate chains. AF-28 immunoblotting of synovial fluid aggrecan fragments revealed a population of small AF-28 fragments of 30-50 kD. Based on their size relative to characterized products of an MMP-8 digest (Fosang, A.J., K. Last, P. Gardiner, D.C. Jackson, and L. Brown. 1995, Biochem. J. 310:337-343), these AF-28 fragments were derived from proteinase cleavage at, or near, the ...ITEGE373 / ARGSV... aggrecanase site. Immunodetection with polyclonal anti-ITEGE antiserum revealed that these fragments lacked the ...ITEGE374 COOH terminus and were not therefore products of aggrecanase action. The same fluid samples contained a broad 68-90-kD G1 fragment that contained the COOH-terminal ...ITEGE374 neoepitope. The results suggest that in some circumstances, despite extensive proteolysis of the core protein, aggrecan molecules may be cleaved by MMPs or aggrecanase in the interglobular domain, but not both.

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.

Cartilage degradation is fully reversible in the presence of aggrecanase but not matrix metalloproteinase activity

IntroductionPhysiological and pathophysiological cartilage turnover may coexist in articular cartilage. The distinct enzymatic processes leading to irreversible cartilage damage, compared with those needed for continuous self-repair and regeneration, remain to be identified. We investigated the capacity of repair of chondrocytes by analyzing their ability to initiate an anabolic response subsequent to three different levels of catabolic stimulation.MethodsCartilage degradation was induced by oncostatin M and tumour necrosis factor in articular cartilage explants for 7, 11, or 17 days. The catabolic period was followed by 2 weeks of anabolic stimulation (insulin growth factor-I). Cartilage formation was assessed by collagen type II formation (PIINP). Cartilage degradation was measured by matrix metalloproteinase (MMP) mediated type II collagen degradation (CTX-II), and MMP and aggrecanase mediated aggrecan degradation by detecting the 342FFGVG and 374ARGSV neoepitopes. Proteoglycan turnover, content, and localization were assessed by Alcian blue.ResultsCatabolic stimulation resulted in increased levels of cartilage degradation, with maximal levels of 374ARGSV (20-fold induction), CTX-II (150-fold induction), and 342FFGVG (30-fold induction) (P < 0.01). Highly distinct protease activities were found with aggrecanase-mediated aggrecan degradation at early stages, whereas MMP-mediated aggrecan and collagen degradation occurred during later stages. Anabolic treatment increased proteoglycan content at all time points (maximally, 250%; P < 0.001). By histology, we found a complete replenishment of glycosaminoglycan at early time points and pericellular localization at an intermediate time point. In contrast, only significantly increased collagen type II formation (200%; P < 0.01) was observed at early time points.ConclusionCartilage degradation was completely reversible in the presence of high levels of aggrecanase-mediated aggrecan degradation. After induction of MMP-mediated aggrecan and collagen type II degradation, the chondrocytes had impaired repair capacity.

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.

Matrix metalloproteinases 19 and 20 cleave aggrecan and cartilage oligomeric matrix protein (COMP)

Matrix metalloproteinase (MMP)‐19 and MMP‐20 (enamelysin) are two recently discovered members of the MMP family. These enzymes are involved in the degradation of the various components of the extracellular matrix (ECM) during development, haemostasis and pathological conditions. Whereas MMP‐19 mRNA is found widely expressed in body tissues, including the synovium of normal and rheumatoid arthritic patients, MMP‐20 expression is restricted to the enamel organ. In this study we investigated the ability of MMP‐19 and MMP‐20 to cleave two of the macromolecules characterising the cartilage ECM, namely aggrecan and the cartilage oligomeric matrix protein (COMP). Both MMPs hydrolysed aggrecan efficiently at the well‐described MMP cleavage site between residues Asn341 and Phe342, as shown by Western blotting using neo‐epitope antibodies. Furthermore, the two enzymes cleaved COMP in a distinctive manner, generating a major proteolytic product of 60 kDa. Our results suggest that MMP‐19 may participate in the degradation of aggrecan and COMP in arthritic disease, whereas MMP‐20, due to its unique expression pattern, may primarily be involved in the turnover of these molecules during tooth development.