Proteases are responsible for enzymatic cleavage of peptide bonds 12, which is a basic requirement for completion of diverse biological processes. Examples of contributions made by proteases can be found in digestion, blood coagulation and fibrinolysis. They are also involved in the processing of precursors related to the synthesis of collagen, immune functions, development and apoptosis 3. The proteolytic activity of proteases must be rigorously controlled to avoid inappropriate degradation of proteins. Imbalance in regulation of proteolytic activity can be found in a wide range of diseases, including cancer, rheumatoid arthritis (RA) and osteoarthritis (OA) 4.

Of particular importance is that proteases have been found to play diverse and strategic roles in cartilage and bone remodelling, which in recent years has engendered increased interest in these enzymes in the field of rheumatology. To highlight the clinical relevance of proteinases to joint destruction, we discuss their contribution to cartilage and bone homeostasis in health and give particular emphasis to their crucial role in diseases such as RA, OA and spondyloarthritis.

Proteases selectively hydrolyze a peptide bond in a polypeptide chain of a target molecule. Depending on the position of the peptide bond, proteases are referred to as exopeptidases or endopeptidases. Exopeptidases specifically cleave substrates at the amino-terminal or carboxyl-terminal positions of polypeptides, and therefore can be subdivided into aminopeptidases and carboxypeptidases 56. Endopeptidases (also called proteinases) break peptide bonds in the middle of the molecule. They can be subclassified based on their mechanism of catalysis, which is related to the chemical group involved in the process of hydrolysis. As a consequence, endopeptidases are described as aspartate, cysteine and threonine types, which act intracellularly in an acid pH, or as serine and metallo catalytic types, which act extracellularly in a neutral pH environment 6. Each of these catalytic types is described in the following discussion (a summary is provided in Figure 1).

The MMPs and adamalysins are considered to be major mediators of cartilage destruction in arthritic diseases and have attracted particular research interest.

It is generally accepted that proteolytic enzymes are involved in the catabolic aspect of normal tissue remodelling and that altered activity of these enzymes is responsible for the cartilage destruction and bone erosion associated with disorders such as OA and RA 30.

Articular cartilage in adults is a comparatively acellular tissue, with a cell volume approximating 2% of the total cartilage volume. The remainder is occupied by an extensive ECM 31. The structural backbone of this matrix is the collagen fibril. Articular cartilage is mainly composed by type II collagen, but it also contains types IX and XI collagen, both on the surface and within. Many other matrix molecules are found in association with the collagen fibrils, the most common and largest of which is the large proteoglycan aggrecan. It forms molecular aggregates with hyaluronic acid, which in turn interacts with the collagen fibrillar network 32. Further elements of the cartilage matrix are leucine-rich proteoglycans, including decorin, fibromodulin and biglycan 32.

Cartilage and bone turnover is a complex process in which proteinases play a prominent role in health and disease. The cartilage remodelling process is conducted entirely by a single cell type, namely the chondrocyte. This cell is not only responsible for the synthesis of the complex ECM of the articular cartilage, but it is also the source of proteinases and other mediators that degrade the damaged matrix to permit repair 33. The production of these proteolytic enzymes is regulated by various local mediators, such as cytokines, growth factors, prostaglandins, matrix breakdown products, complement, oxygen species and neuropeptides 34.

Similar to cartilage, the bone tissue undergoes continuous remodelling. Osteoclasts play a prominent role in the resorption of bone in this remodelling cycle. By secreting H⁺ ions and proteinases, osteoclasts dissolve bone mineral and degrade organic bone matrix 35. Osteoclast-mediated bone resorption is a multistep process that is initiated by the attachment of osteoclasts to the bone surface. After they have attached to the bone surface, a tightly sealed resorption lacuna is created. Then proteolytic enzymes expressed in osteoclasts, such as cathepsin K and MMPs (MMP-13, MMP-2 and MMP-9), are secreted into the lacuna for removal of bone mineral and degradation of organic matrix protein 32. MMPs are essential for the initiation of the osteoclastic resorption process by removing the collagenous layer from the bone surface, which must be achieved before the demineralization process can be initiated. MMPs have also been implicated in the cleansing of bone lining cells from resorption pits of remaining collagen fibrils before the pits are refilled with new bone matrix components produced by osteoblasts 11. Additionally, MMP-12 produced by osteoclasts or periosteoclastic cells contributes to bone matrix solubilization and osteoclast migration, thereby controlling the cell-matrix interactions that are required for cell attachment/detachment 36. In summary, proteinases participate importantly in normal bone and cartilage turnover, whereas deregulation of proteinases is relevant to several joint diseases.

The cellular and molecular mechanisms that underlie pathological bone destruction have partially been identified. In particular, molecular insight into osteoclast biology has revealed that even though inflammation and destruction are independent processes, they are linked to each other 30. The link is established through the network of cytokines and growth factors that are produced by cells in the inflamed joint 35. Proinflammatory cytokines such as TNF-α and IL-1 are abundant in the synovium of patients with various types of chronic arthritis, and they disrupt normal tissue homeostasis in cartilage and bone 30. The influence of cytokines in osteoclast differentiation provides a link between inflammation and the process of bone destruction. Some cytokines such as IL-1 directly induce osteoclast differentiation, whereas others such as TNF-α act indirectly by upregulating RANKL (receptor activator of nuclear factor-κB ligand), which is the major factor involved in osteoclast differentiation and activation 37.

One of the strongest predictors of long-term outcome in RA and OA is progressive joint damage. In RA and OA, this progressive cartilage and bone destruction are considered to be driven by excess MMP enzymes 38. The profile of MMPs expressed by activated cells in arthritic joints is sufficient to destroy completely the structural collagens that build up the articular cartilage, the adjacent bones and tendons, as well as the noncollagen matrix molecules 7.

In RA functional disability is a multifactorial process, and damage to the joint structures contributes significantly to the overall functional status of the patient 31. Degradation of the cartilage, tendon and bone ECM proteins by MMPs is the hallmark of synovial joint destruction 39. In this process loss of aggrecans, considered a critical event in arthritis, initially occurs at the surface of the cartilage and then progresses to deeper zones. This is followed by degradation of collagen fibrils and mechanical failure of the tissue 39.

There are two principal mechanisms by which RA synovial tissue contributes to loss of cartilage. The first and direct mechanism involves the production of MMPs and cathepsins by the RA synovium 33. The second mechanism indirectly induces cartilage remodelling by deregulation of chondrocyte function through the release of cytokines and other mediators from the synovium 33. As part of the inflammatory process in RA, macrophages are recruited to the joints, where they release inflammatory cytokines such as IL-1β, TNF-α and IL-6. These cytokines induce expression of proteinases in synovial fibroblasts and chondrocytes 40.

It has been demonstrated that synovial cells stimulated by TNF-α or IL-1β increase the transcription of cathepsin B 404142. The location of cathepsin B appeared to be restricted to synovial cells attached to cartilage and bone at sites of RA joint erosion 43. Accumulation of active cathepsin B in the synovial fluid of RA patients is probably related to destruction of subchondral bone 44. Interestingly, cathepsin B, along with MMP-1 and cathepsin L, has been detected in the synovium soon after onset of symptoms, implying that the potential for joint destruction exists even at very early stages of the disease 4546.

Also, cathepsin K is highly expressed in synovial fibroblasts and macrophages in joints of RA patients 114748. In addition to its major role in bone resorption, it has been demonstrated that cathepsin K plays a critical role in cartilage degradation as well. Co-cultures of synovial fibroblasts on cartilage disks revealed the ability of fibroblasts to phagocytose collagen fibrils 49. This degenerative property of fibroblasts was prevented by a potent cathepsin K inhibitor.

The important role of MMPs in cartilage and bone destruction in RA has been demonstrated using various approaches. High levels of MMP-1, MMP-3, MMP-8 and MMP-9 are found in the synovial fluid of patients with RA 50. Moreover, the synovial tissue exhibits high constitutive expression of MMP-2, MMP-11 and MMP-19 51. The expression of MMP-3 is particularly high in synovial tissue from RA patients, suggesting that MMP-3 plays a significant role in the progression of erosions of the cartilage 52. Interestingly, a high MMP/TIMP ratio was identified in the serum of RA patients 52, implying an imbalance in the proteolytic system in favour of the MMPs.

Finally, it has been shown that RA synovial fibroblasts exhibit increased production of MMPs (MMP-1, MMP-3, MMP-13, MMP-14 and MMP-15) and contribute significantly to the joint destruction observed in RA 5354. This high expression of MMPs by RA synovial fibroblasts is not only upregulated by elevated levels of IL-1β and TNF-α 40 but is also sustained intrinsically by RA synovial fibroblasts, which display a transformed phenotype 55.

In summary, the inflammatory environment observed in the synovial tissue allows the production and secretion of cytokines and growth factors by infiltrating cells and resident synovial cells. This leads to increased production of ADAMTSs and MMPs by synovial fibroblasts and by chondrocytes, favouring cartilage and bone destruction (Figure 2b).

Because of the significant roles played by MMPs in joint destruction, they have been regarded as useful biomarkers and therapeutic targets. Levels of MMP-1 and MMP-3 in the serum of RA patients correlate with disease activity 56. For that reason, it has been suggested that MMP-3 could be a useful marker for predicting of bone and cartilage damage in early untreated RA 57. Successful treatment with leflunomide 58, methotrexate 53, or anti-TNF-α antibodies efficiently downregulates serum levels of MMPs 5960. Although these recent advances in RA treatment arrest radiological joint destruction for some time, none of the disease-modifying antirheumatic dugs, including the biological agents, have yet provided long-term, problem-free protection against joint destruction 54. Therefore, there remains a need to develop novel therapeutic strategies.

The aetiology of OA is not completely understood, but it appears to result from mechanical, biochemical and enzymatic factors. The final common pathway of these interactions is the failure of the chondrocytes to maintain a homeostatic balance between matrix synthesis and degradation 1961. Therefore, excessive digestion of cartilage collagen is considered a critical issue in loss of articular cartilage in OA 62. However, the chondrocytes not only secrete the proteinases responsible of cartilage destruction, but they also produce proinflammatory cytokines such as IL-1 and TNF-α, creating an inflammatory environment that also favours increased synthesis of proteinases 17 (Figure 2a).

The destruction of the ECM in OA results from several events that take place in sequence. The first critical step is loss of cartilage aggrecans mediated by ADAMTS-4 and ADAMTS-5. Then, diverse MMPs continue to degrade the major components of the ECM 63. Secondary cartilage breakdown products, such as fibronectin fragments, are released into the joint fluid and irritate the synovial membrane lining in the joint space. The resulting synovitis provokes release of inflammatory mediators from synovial tissue and initiates recruitment of mononuclear inflammatory cells into the joint space. These arriving cells secrete IL-1 and TNF-α, which further upregulate the production of proteinases by chondrocytes and synovial fibroblasts 6465 (Figure 2).

In OA, expression of MMPs, ADAMTS and TIMPs exhibits a particular pattern 66. The coexistence of multiple collagenases, plus their distinct localization and distribution in the cartilage, points to a specific role for each of them. For example, it is proposed that MMP-1 is involved in tissue destruction initiated in the superficial zone of the cartilage during inflammation, whereas MMP-13 plays a role in the remodelling phase of the disease 19. Furthermore, it has been shown that MMP-2 and MMP-9 are increased in OA joints and that their expression is enhanced by IL-1 and TNF-α 19. In OA, TIMPs do not increase to the same extent as proteinases do, producing a disproportionate ratio of MMP to TIMP. The result is an excess of proteinases, which aggravates cartilage breakdown and promotes joint destruction 6667.

The spondyloarthropathies (SpAs) represent a group of related arthritides that include ankylosing spondylitis, psoriatic arthritis, reactive arthritis, and enteropathic and undifferentiated SpA. They are characterized by their association with HLA-B27 and development of sacroilitis and enthesitis. The observed functional impairment, disability and impaired quality of life resemble those in RA 68.

MMPs and TIMPs are expressed in the synovial compartment of SpA patients 67. In particular, high expression of MMP-3 has been demonstrated in serum, synovial tissue and synovial fluids of SpA patients 6970. TNF-α blockade can induce a downregulation of MMPs and TIMPs in the synovium of affected joints and decreases levels of MMP-3 in the serum 70. Consequently, some authors have predicted a possible role for MMPs as biomarkers of disease activity or response to treatment 71.

Most recently, our group showed that new small erosions appear in the bony processes of the vertebrae even in late stages of the disease (at the time of surgical correction), and that these sites of destruction appear to be caused by osteoclasts producing MMPs, in particular cathepsin K 72. These findings might explain why patients still experience significant pain at late stages of the disease.

Because of the prominent roles of metalloproteinases observed in degenerative diseases, they are attractive as therapeutic targets. Several approaches to block the deleterious effects of proteinases in pathological conditions have been evaluated. These approaches include use of synthetic metalloproteinase inhibitors, inhibition of signal transduction pathways and gene transfer technology.

Tight control of proteinases in the joints allows the integrity of the bone and cartilage to be conserved. Failure to regulate the synthesis, activation and inhibition of the proteinases favours the deleterious effects of MMPs, including MMP-1, MMP-3 and others, which finally lead to degradation. Despite early disappointing results with synthetic inhibitors of MMPs in human trials, there remains much scope for developing effective but highly selective and safe MMP inhibitors. This novel group of drugs could provide new therapeutic options to inhibit joint destruction, which is the main reason for the disability observed in RA, OA and the SpAs.

ADAM = a disintegrin and metalloproteinase; ADAMTS = a disintegrin and metalloproteinase with thrombospondin motif; ECM = extracellular matrix; IL = interleukin; MMP = matrix metalloproteinase; MT = membrane-type; OA = osteoarthritis; RA = rheumatoid arthritis; SpA = spondy-loarthropathy; TIMP = tissue inhibitor of metalloproteinases; TNF = tumour necrosis factor; uPA = urokinase-type plasminogen activator.

The authors declare that they have received a research grant to test different compounds from Novartis, which are not mentioned in this review.