The role of aggrecan in normal and osteoarthritic cartilage
© Roughley and Mort; licensee Springer 2014
Received: 12 May 2014
Accepted: 22 May 2014
Published: 16 July 2014
Aggrecan is a large proteoglycan bearing numerous chondroitin sulfate and keratan sulfate chains that endow articular cartilage with its ability to withstand compressive loads. It is present in the extracellular matrix in the form of proteoglycan aggregates, in which many aggrecan molecules interact with hyaluronan and a link protein stabilizes each interaction. Aggrecan structure is not constant throughout life, but changes due to both synthetic and degradative events. Changes due to synthesis alter the structure of the chondroitin sulfate and keratan sulfate chains, whereas those due to degradation cause cleavage of all components of the aggregate. These latter changes can be viewed as being detrimental to cartilage function and are enhanced in osteoarthritic cartilage, resulting in aggrecan depletion and predisposing to cartilage erosion. Matrix metalloproteinases and aggrecanases play a major role in aggrecan degradation and their production is upregulated by mediators associated with joint inflammation and overloading. The presence of increased levels of aggrecan fragments in synovial fluid has been used as a marker of ongoing cartilage destruction in osteoarthritis. During the early stages of osteoarthritis it may be possible to retard the destructive process by enhancing the production of aggrecan and inhibiting its degradation. Aggrecan production also plays a central role in cartilage repair techniques involving stem cell or chondrocyte implantation into lesions. Thus aggrecan participates in both the demise and survival of articular cartilage.
This review describes the role of aggrecan in the function of articular cartilage and how this role is perturbed in the osteoarthritic (OA) joint. It explains the structure/function relationship between the various regions of the aggrecan molecules and how these are altered by synthetic and catabolic events that occur throughout life and in the diseased joint. It is clear that a view of cartilage function based on the contribution of one molecule will be somewhat biased, but it is also arguable that no molecule plays a more important role than aggrecan. Without it the tissue could not withstand the rigours of joint loading and its decreased abundance marks the onset of the tissue decline associated with the OA joint.
Articular cartilage, aggrecan and OA
Articular cartilage is the white, smooth, lustrous connective tissue that covers the surfaces of bones where they meet in diarthrodial joints. It serves two major functions; to provide almost frictionless motion and to counteract the impact of the compressive forces experienced across the joint during use. The first of these functions is not usually associated with aggrecan, but relates to the unique collagen fiber organization at the articular surface and the presence of lubricin and hyaluronan (HA)/phospholipid complexes that aid in joint lubrication (Gleghorn et al. ; Wang et al. [2013a]). However, aggrecan does play a role in fluid pressurization of the cartilage which supports the articular surface and so may facilitate its function (Dabiri and Li, ; Moore and Burris, ). The ability to resist compression is intimately associated with the high abundance of aggrecan throughout the extracellular matrix of articular cartilage beneath its superficial zone.
The OA joint is characterized by catabolic processes that degrade both the HA backbone of the aggregate and the core protein of the aggrecan molecules, so impairing aggrecan function and predisposing the articular cartilage to erosion. Such degradation is associated with proteinases, hyaluronidases and free radicals.
Structure of aggrecan
The G2 and G3 regions are separated by a long GAG-attachment region that is subdivided into three domains (Doege et al. ). The KS-rich domain lies adjacent to the G2 region. It is composed of repeats of six amino acids, with each repeat possessing a proline-serine sequence. Each serine residue can potentially act as the attachment site for O-linked oligosaccharides that form the linkage region for KS. The KS-rich domain is followed by the chondroitin sulfate (CS)-rich domain, which is divided into two subdomains (CS1 and CS2) that differ in amino acid sequence. The CS1 domain is composed of repeats of nineteen amino acids, with each repeat possessing two serine-glycine sequences. Each serine residue in the repeats can potentially act as an attachment site for CS. The CS2 domain also possesses serine-glycine sequences that can act as the attachment site for CS. Because of their differing amino acid sequences, the CS1 and CS2 domains have differing susceptibilities to proteolysis. A typical aggrecan molecule may contain up to 100 CS chains and a lower number of KS chains. The sulfated nature of the CS and KS provide the aggrecan with its high anionic charge.
The CS2 domain is followed by the G3 region, which resides at the carboxy terminus of the core protein. The G3 region is composed of two epidermal growth factor (EGF)-like domains, one C-type lectin-like domain, and one complement regulatory protein (CRP)-like domain. The G3 region appears to be essential for normal trafficking of the aggrecan within the chondrocyte and for its secretion into the extracellular matrix (ECM) (Zheng et al. ). It is unclear whether each domain of the G3 region possesses a unique function within the ECM. The lectin-like domain does have the ability to interact with a variety of ECM proteins, such as tenascins and fibulins (Aspberg, ), and could potentially play a role in anchoring the aggrecan within the tissue. However, the G3 region is absent from many mature aggrecan molecules in the ECM due to its proteolytic cleavage (Dudhia et al. [1996a]).
The domain structure of the aggrecan core protein is reflected in the exon organization of its gene (Valhmu et al.) (Figure 2). The human aggrecan gene consists of 19 exons. The G1 region is encoded by exons 3–6, with exon 3 encoding the A domain, exons 4 and 5 encoding the B domain and exon 6 encoding the B′ domain. The IGD is encoded by exon 7. The G2 region is encoded by exons 8–10, with exons 8 and 9 encoding the B domain and exon 10 encoding the B′ domain. The GAG attachment region is encoded by exons 11 and 12, with exon 11 encoding the first part of the KS-rich domain, and the large exon 12 encoding the remainder of the KS-rich domain plus the CS1 and CS2 domains. The hexapeptide repeats of the KS-rich region are encoded by exon 12. The G3 region is encoded by exons 13–19, with exons 13 and 14 each encoding an EGF-like domain, exons 15–17 encoding the lectin-like domain, and exon 18 encoding the CRP-like domain.
While the domain organization of aggrecan is conserved amongst different species, there are species variations in its core protein structure (Doege et al. ; Hering et al. ; Li et al. ; Walcz et al. ). Foremost amongst these are the variations in the number of repeats in both the KS-rich and CS domains. The number of KS-rich repeats varies from 4 in the rat to 23 in the bovine, with the human possessing 13 (Barry et al. ). In the CS1 domain there are 17 repeats in the rat, 23 in the mouse and 27 in the bovine. The human is unique in possessing length polymorphism in its CS1 domain, with the number of repeats varying from 13 to 33, though the majority of individuals have 26–28 repeats (Doege et al. ). In the human it has been shown that the KS-rich domain is devoid of CS and the CS1 domain is devoid of KS (Rodriguez et al. ). The human CS2 domain contains at least one KS chain adjacent to the G3 region. In the bovine this site is occupied by CS. The human also appears to be unique in possessing two exons encoding for EGF-like domains. In other species studied, genomic sequence variations prevent expression of the EGF1 domain and only the EGF2 domain is present (Fülöp et al. ). In the human, the G3 region has a variable structure due to alternative splicing of both EGF-like domains and the CRP-like domain (Grover and Roughley, ).
The HA that forms the core of the proteoglycan aggregate is not unique to cartilage, but is ubiquitous in its presence (Fraser et al. ). It is formed at the plasma membrane of most cells by a hyaluronan synthase (Has) (Weigel and DeAngelis, ). Mammals possess 3 Has (Has1, Has2 and Has3) (Itano et al. ), with Has2 being the predominant form in cartilage (Recklies et al. ). The HA is synthesized in the cytosol and the growing chain is extruded directly into the extracellular environment (Hubbard et al. ), where it forms a coat around the chondrocytes (Knudson et al. ). It is not clear how HA is released from the cell surface or where interaction with aggrecan takes place. A typical HA chain may possess 10,000 disaccharide units, with each G1 domain interacting with a decasaccharide region (Hascall and Heinegård, ). Individual aggrecan molecules may be spaced about 50 disaccharides apart due to the size exclusion properties of their GAGs.
Structure/function relationships of aggrecan
The movement of water within the cartilage during cyclical compressive loading is thought to aid in the nutrition of the chondrocytes. As the cartilage is an avascular tissue, its nutrients must arise from the bathing synovial fluid by diffusion and its waste products must be expelled in a similar manner. The displacement of water upon compression aids in waste removal, and the return of water following removal of compression aids in nutrient supply. Obviously static loading cannot participate in such a mechanism. However, while dynamic loading may be beneficial to cartilage, its value will depend on its magnitude, frequency and duration. Loading at too high a magnitude may adversely influence the cells, resulting in cell death and ECM destruction. Cartilage overloading has long been thought to be linked to the onset of OA (Mitchell and Cruess, ).
Variations in aggrecan structure due to synthesis
Variations in aggregate structure due degradation
The major changes in aggregate structure taking place in the ECM are due to degradation, and may affect all components of the aggregate (Figure 6). The protein/carbohydrate makeup of the proteoglycan aggregate renders it susceptible to multiple degradative mechanisms. Most importantly the protein component can be cleaved by a diverse series of proteolytic enzymes and the glycosaminoglycan component, in particular the HA, is susceptible to the action of glycosyl hydrolases. In addition, both the protein and polysaccharide constituents undergo modification by reactive oxygen species (ROS) which can lead to chain fragmentation.
Sequence termini on cleavage fragments of human aggrecan for which anti-neoepitope antibody reactivity, or direct sequencing, have demonstrated the presence of specific products in vivo
(Flannery et al. )
(Fosang et al. )
(Chamberland et al. )
(Lark et al. )
(Sandy et al. )
(Maehara et al. )
(Struglics et al. )
(Sandy and Verscharen, )
(Sandy and Verscharen, )
(Dufield et al. )
(Sandy and Verscharen, )
In addition to the metalloproteases, there is direct evidence for the involvement of specific cysteine and serine proteases in cartilage turnover. The presence of m-calpain in human cartilage has been reported for some time and it is clear that much of the fragmented aggrecan present in adult human cartilage, which consists of the G1-IGD-G2 regions, is the product of calpain action (Maehara et al. ). Calpain is a multidomain calcium dependent protease and would be active in the cartilage extracellular matrix. There is accumulating evidence that in addition to the action of MMP collagenases, the cysteine protease cathepsin K also plays a role in collagen breakdown in human cartilage. In contrast to the other proteases considered so far, cathepsin K consists of a single catalytic unit but forms a defined complex with chondroitin 4-sulfate which increases its stability and is essential for its ability to degrade triple helical collagen. Since cathepsin K is known to cleave aggrecan in vitro (Hou et al. ), it is likely to contribute to this process in vivo.
Recently direct analysis of human osteoarthritic cartilage extracts identified the interesting serine protease HtrA1 and aggrecan degradation products characteristic of its action in the IGD (Chamberland et al. ). Since elevated levels of this protease are seen in osteoarthritis (Grau et al. ) and given its ability to cleave many other extracellular matrix components (Eigenbrot et al. ), it may complement the well accepted role of the metalloproteases in disease progression.
While there is little evidence for degradation of CS and KS within the ECM, the size of the HA decreases with age (Holmes et al. ), presumably due to depolymerization by hyaluronidases or free radicals (Stern et al. ). A series of genes have been identified coding for glycosyl hydrolase-like proteins termed hyaluronidases (Csoka et al. ; Stern et al. ). However only two of these appear to be true hyaluronidases relevant to extracellular matrix degradation. While hyaluronidase-1 cleaves hyaluronan and chondroitin sulfate very effectively, it has a very low pH optimum as befits its normal lysosomal location. In contrast hyaluronidase-2 is active at neutral pH and at least a portion of it is located at the cell surface (Miller et al. ). Hyaluronidase-2 has been shown to cleave hyaluronan into relatively large fragments which are then taken up by the cell and degraded by hyaluronidase-1 and β-hexosaminidase (Gushulak et al. ).
Proteolysis causes extensive modification of the aggrecan core protein. This involves removal of the G3 region (Dudhia et al. [1996b]), truncation within the GAG-attachment region and ultimately accumulation of G1 domains by cleavage within the IGD (Roughley et al. ) (Figure 5). In the case of link protein, proteolysis results in the accumulation of LP3 and a concomitant decrease in LP1 and LP2. In vitro, many proteinases are capable of forming LP3, but in vivo MMPs play a major role (Nguyen et al. ). Aggrecanases are not able to cleave link protein (Roughley et al. ). While cleavage of link protein may be of no functional consequence, cleavage of aggrecan and HA are detrimental to cartilage function as they reduce both the size and charge of the aggregates.
Variations in aggrecan structure in OA
Articular cartilage in the OA joint is subjected to increased catabolism due to the presence of various cytokines that may arise from the chondrocytes themselves or by infiltration from the inflamed synovium (Goldring and Goldring, ). Foremost amongst these cytokines are interleukin 1 (IL1) and tumor necrosis factor α (TNFα), which not only stimulate the production of proteinases but also down-regulate aggrecan production. These inflammatory cytokines are associated with the production of aggrecanases and MMPs, which degrade the aggrecan core protein (Troeberg and Nagase, ). The combined effect of increased degradation coupled with decreased synthesis results in aggrecan loss from the ECM and impairment of articular cartilage function. Subsequently, collagenases (MMP1 and MMP13) degrade the collagen fibrils of the tissue, initiating tissue fibrillation and eventual erosion. Even if joint inflammation is resolved, it is likely that the depletion in aggrecan results in adverse consequences to loading, with the production of proteinases by the chondrocytes. The initiating event that causes aggrecan depletion may be either joint inflammation or joint overloading. Once damage is initiated, articular cartilage has little ability for endogenous repair and further damage appears to be an inevitable consequence.
While ineffective, articular cartilage does mount a limited repair response characterized by some cell proliferation and some new proteoglycan synthesis around the resulting cell clusters. Analysis of the aggrecan from OA cartilage shows a CS sulfation pattern more typical of juvenile cartilage than the mature adult (Caterson et al. ), suggesting that OA alters the phenotype of the resident mature chondrocytes or that it induces the formation of new immature chondrocytes. As articular cartilage is known to possess progenitor cells (Williams et al. ), these could be the source for cell proliferation and differentiation into immature chondrocytes.
Retention and loss of aggrecan
Following proteolytic cleavage of the aggrecan core protein at any site, two fragments are generated. One fragment will possess a G1 region and remain bound to HA, whereas the other is no longer able to interact with HA. This latter non-aggregating fragment is free to diffuse within the ECM and is rapidly lost into the synovial fluid. In contrast, fragments that remain bound to HA can reside in the tissue for many years (Maroudas et al. ). It has been estimated that the G1 regions that form the end product of proteolytic truncation of the aggrecan core protein have a half-life of about 20 years. This can be viewed as an impediment to repair, as the G1 regions cannot participate in the load bearing function of the cartilage, yet they occupy space on the HA that could be used for binding newly synthesized aggrecan.
The aggrecan fragments that diffuse into the synovial fluid have been used as biomarkers in OA patients, with the premise that a higher concentration of fragments in the synovial fluid reflects enhanced degradation within the cartilage. This does assume that other factors, such as the rate of clearance from the synovial fluid or the secretion of newly synthesized aggrecan from the cartilage, are of lesser importance. In principle, the efficacy of drug therapy can be followed by monitoring the decline in fragment abundance. Fragment abundance can be monitored by a variety of techniques. The simplest techniques involve the quantification of sulfated GAG content by the dimethyl methylene blue (DMMB) assay (Farndale et al. ) and analysis of KS by immunoassay (Campion et al. ). An immune assay has also been described for a CS sulfation variant (846 epitope) that is characteristic of juvenile and OA cartilage (Poole et al. ). Finally, neoepitope immunoassays have been developed to specifically analyse for aggrecan degradation products resulting from either MMP or aggrecanase cleavage (Fosang et al. ; Germaschewski et al. ; Hughes et al. ).
Repair of articular cartilage
While joint replacement with a prosthesis may be the preferred treatment for late stage OA where most of the articular cartilage has been eroded, biological repair is an attractive alternative for more focal early lesions. Current techniques tend to fall into two categories: those that are cartilage-based and those that are cell-based (Camp et al. ). The cartilage-based procedures include mosaicplasty, single osteochondral autografts and osteochondral allografts, where a mature articular cartilage is implanted into the lesion so that the composition of the repair tissue is equivalent to that which was eroded. The cell-based techniques include microfracture and autologous chondrocyte implantation (ACI), where the cells that will generate the repair cartilage are either bone marrow-derived mesenchymal stem cells or mature articular chondrocytes, respectively. When using stem cells, the structure and abundance of aggrecan and the composition of the cartilage ECM that is formed is immature and may not be ideal for withstanding the rigours of adult life. The use of mature chondrocytes may help resolve some of these problems, but the cartilage structure is still somewhat immature as age-dependent events, such as collagen cross-linking, that help reinforce the tissue are not fully established. A means of maturing the cell-derived cartilage would enhance its repair value. However, whichever technique is used, it is essential that the factor causing the degeneration be treated or eliminated if future degeneration of the repair tissue is to be avoided (Moran et al. ).
In the early stages of OA, prior to extensive collagen damage, it may be possible to retard or even reverse the degenerative process by the administration of agents that promote aggrecan synthesis and prevent its degradation. Development of specific small molecule inhibitors for MMP and ADAMTS family members has been an extremely active area for several decades. Although there have been setbacks (Clark and Parker, ), new highly specific compounds for the inhibition of ADAMTS5 are being produced (Deng et al. ) so there is still hope that products of this approach may reach the clinic. Nutriceuticals, such as glucosamine, have been touted as therapeutic agents, though their clinical efficacy remains to be proven (Salazar et al.). Some growth factors, such as transforming growth factor β (TGFβ) and bone morphogenetic protein 7 (BMP7) have the desired properties, but they are expensive to use and could have unwanted side effects. However, local delivery of single or a combination of growth factors, including insulin-like growth factor I (IGFI), fibroblast growth factor-2 (FGF2), TGFβ and BMPs, by way of gene therapy may overcome these deficits (Shi et al. ; Trippel et al. ). An economically attractive alternative to growth factors may be LinkN (McKenna et al. ), which represents the amino terminal 16 amino acids of link protein. It occurs naturally from the formation of LP3, but can be produced cheaply using a peptide synthesizer. It appears to function through the BMP receptor system (Wang et al. [2013b]), but unlike BMPs does not stimulate osteogenesis (Antoniou et al. ). Only in the future will we know whether such agents live up to their current promise.
Aggrecan plays an essential role in the function of articular cartilage and is a central participant in its destruction in the OA joint. Agents that prevent aggrecan degradation and restore its production may play a future role in the treatment of early stage OA.
The authors would like to thank Guylaine Bédard for the artwork in the figures.
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