Loss of articular cartilage is a central feature of both osteoarthritis (OA) and rheumatoid arthritis (RA). Cartilage destruction in both diseases is associated with increased production of proteinases, which may be driven by inflammatory cytokines such as interleukin-1 (IL-1), tumor necrosis factor (TNF), and IL-17. The evidence is strong that cartilage resorption is caused by inflammatory cytokines in RA, since they are known to be produced in the diseased joint, and anticytokine therapy has beneficial effects (1). The nature of the stimulus causing chondrocytes to catabolize their matrix in OA is obscure. There is some evidence that the chondrocytes themselves may express IL-1 (2), but the cause and pathophysiologic significance of this are unknown.
Cartilage is made of soluble polymers trapped in a mesh of insoluble fibers largely comprising type II collagen. There are 2 stages in cartilage resorption. First, there is loss of aggrecan, the major soluble component, and this is followed by loss of collagen. The first step is potentially reversible, since mature chondrocytes can resynthesize the aggrecan. The second step is largely irreversible because the chondrocytes of mature articular cartilage are unable to restore the original fibrous network (3).
Aggrecan is a proteoglycan that forms very large aggregates by binding to hyaluronic acid. It is heavily substituted with sulfated glycosaminoglycan (GAG) chains that retain the water that enables cartilage to resist compression. Aggrecan is lost as a result of proteolysis, particularly that due to aggrecanases that cleave the core protein at characteristic sites. The C-terminal fragments passively escape from cartilage, while the N-terminal portions may remain associated with the hyaluronic acid. Following the loss of aggrecan, the collagen fibers are exposed to attack by specific collagenases, such as matrix metalloproteinase 1 (MMP-1) and MMP-13 (4).
The mechanisms by which IL-1 causes cartilage destruction have been investigated in both cell and organ culture. IL-1 increases chondrocyte aggrecanase activity (5–7). The proteinases that carry out the specific cleavages belong to the ADAMTS class of enzymes. ADAMTS-1 (8), ADAMTS-4 (9), ADAMTS-5 (10), ADAMTS-8 (11), and ADAMTS-9 (12) all cleave aggrecan at the specific sites. IL-1 increases expression of ADAMTS-4 and ADAMTS-5 messenger RNA (mRNA) in animal chondrocytes (13, 14), although in human cells, it has been reported to increase only ADAMTS-4 (15–17). ADAMTS-4–knockout mice were found to have no obvious skeletal phenotype; IL-1 caused proteoglycan degradation in their articular cartilage, and the course of a surgically induced model of OA was the same as in normal animals (18). However, the cartilage of ADAMTS-5–knockout mice was resistant to IL-1, and the cartilage proteoglycan was preserved in both RA and OA models (19, 20). Thus, in mice, ADAMTS-5 is strongly implicated as being important in the catabolism of cartilage aggrecan.
In addition to increasing aggrecanase activity, IL-1 also increases expression of several MMPs, including the specific collagenases MMP-1 and MMP-13 (21). MMP-13 has been implicated in cartilage collagenolysis because it preferentially cleaves type II collagen over types I and III collagen (22), and its expression and production are significantly elevated in human OA cartilage (23). While cartilage catabolism has been extensively investigated, relatively little attention has been paid to the possible existence of anticatabolic mechanisms in cartilage. Are there intrinsic factors in articular cartilage that oppose catabolic stimuli such as IL-1? There are old reports that insulin-like growth factor (IGF) (24) and transforming growth factor β (25) can counteract proteoglycan breakdown in animal cartilage stimulated with low-dose IL-1 in vitro. However, the significance of these findings as intrinsic anticatabolic mechanisms in human articular cartilage is unclear. IGF-1 and osteogenic protein 1 (OP-1) have also been reported to inhibit some actions of IL-1 in human cartilage (26, 27). The existence of anticatabolic mechanisms is of potential importance. Their impairment could predispose to tissue degeneration, their augmentation could slow degeneration, and understanding them could suggest new approaches to therapy, particularly for degenerative disease.
We have found that articular chondrocytes are surrounded by an extracellular pool of fibroblast growth factor 2 (FGF-2) (28). This mediates chondrocyte activation when cartilage is loaded (29) and is rapidly released upon cartilage injury (28). FGF-2 induced the synthesis of a number of proteins in porcine chondrocytes, such as tissue inhibitor of metalloproteinases 1 (TIMP-1) and MMPs 1 and 3 (28). These observations prompted us to examine whether FGF-2 affected the response of human articular cartilage to a well-characterized catabolic stimulus such as IL-1. We report that FGF-2 antagonizes the proteoglycan degradation induced by IL-1 or other catabolic stimuli. We also show that FGF-2 inhibits the up-regulation of ADAMTS-4 and ADAMTS-5 induced by IL-1α in human chondrocytes. These findings suggest that perichondral FGF-2 has an anticatabolic chondroprotective function.
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Our finding that IL-1α induces both ADAMTS-4 and ADAMTS-5 in human articular chondrocytes is in contrast to several other reports, although IL-1α does up-regulate both enzymes in animal cartilage (13, 14). Flannery et al (16) found no increases in ADAMTS-1, ADAMTS-2, ADAMTS-3, ADAMTS-4, and ADAMTS-5 mRNA in human articular cartilage stimulated with IL-1 for 4 days. Bau et al (15) found that ADAMTS-4 mRNA was induced in isolated human chondrocytes stimulated for 3 days with IL-1β, whereas ADAMTS-5 was unaffected. Moulharat et al (17) showed variable expression of ADAMTS-4 in explants or isolated chondrocytes in response to stimulation with IL-1β for 1 or 2 days, while ADAMTS-5 expression was constitutive and was not altered. In all of these studies, ADAMTS-5 mRNA was not measured at times earlier than 24 hours after stimulation. We found that IL-1α increased both ADAMTS-4 and ADAMTS-5 mRNA in human articular chondrocytes, but the effect was moderate and transient, especially in the case of ADAMTS-5.
There is still controversy concerning which enzymes are responsible for the degradation of cartilage aggrecan. In mice, ADAMTS-5 is the main aggrecanase because deletion of ADAMTS-5, but not ADAMTS-1 or ADAMTS-4, protects against the catabolic action of IL-1 and against aggrecan loss in the development of OA and inflammatory arthritis (18–20, 39). However, Song et al (40) reported that in human cartilage, knockdown of ADAMTS-4, ADAMTS-5, or both enzymes by small interfering RNA, attenuated the degradation of aggrecan in human cartilage stimulated with a combination of TNFα and oncostatin M (40). This suggested that both ADAMTS-4 and ADAMTS-5 contribute to aggrecanolysis in human tissue. Although we found that the induction of ADAMTS-5 mRNA by IL-1α in human chondrocytes was moderate and transient, it might be important because ADAMTS-5 has at least 1,000-fold higher specific activity on aggrecan than does ADAMTS-4 (31).
Exogenous FGF-2 inhibited IL-1α induction of aggrecanase-dependent aggrecan degradation in human articular cartilage. The suppression was dose dependent and was observed at an FGF-2 concentration as low as 1 ng/ml. FGF-2 did not act as an IL-1α inhibitor because it did not affect IL-1α induction of MMPs 1, 3, and 13 or of IL-6 and IL-8 in human cartilage. It also inhibited aggrecan degradation induced by TNFα or retinoic acid. These findings suggest that the effect of FGF-2 on the action of the inflammatory cytokines is restricted and may be relatively specific to aggrecan degradation. The growth factor does not appear to be having a general antiinflammatory effect. Interestingly, FGF-2 significantly suppressed proteoglycan synthesis, and it is known to inhibit the anabolic activity of IGF-1 and OP-1 in human articular cartilage (26, 27). FGF-2 potentially induces MMP production in cartilage. For example, Im et al (41) reported that FGF-2 stimulates the production of MMP-13 in human articular chondrocytes, and we have reported that MMPs 1 and 3 are induced by FGF-2 in porcine articular chondrocytes (28). Taken together, the evidence suggests that FGF-2 may have multiple functions in cartilage homeostasis; it inhibits aggrecanolysis, but it may promote collagenolysis and reduce aggrecan synthesis.
Chondrocytes are surrounded by an extracellular pool of FGF-2 that is bound to the heparan sulfate chains of perlecan (42). This pool appears to be sequestered but activates the cells when cartilage is compressed by loading. This sequestered pool does not prevent IL-1 from inducing aggrecan breakdown in the cultured cartilage. The fact that adding exogenous FGF-2 activates chondrocytes (e.g., it increased TIMP-1 production) suggests that the pericellular perlecan may be saturated with FGF-2. Interestingly, sequestered endogenous FGF-2 is released and activates chondrocytes when cartilage is injured (28), perhaps causing an anticatabolic effect.
It was surprising that TIMP-1 inhibited IL-1α–induced aggrecan degradation in human cartilage. TIMP-1 has much weaker inhibitory activity against ADAMTS-4 and ADAMTS-5 than does TIMP-3 (43, 44). Some reports describe an inhibitory effect of TIMP-1 on cartilage degradation. For example, Arner et al (45) reported that aggrecanase activity in conditioned medium from IL-1β–stimulated bovine nasal cartilage was inhibited by bovine TIMP-1 with a 50% inhibitory concentration of 210 nM, while Hughes et al (46) found that 255 nM TIMP-1 partially inhibited aggrecanase activity of conditioned medium from IL-1α–stimulated porcine chondrocytes. Bonassar et al (47) reported that recombinant human TIMP-1 significantly inhibited retinoic acid– or IL-1β–induced aggrecan degradation in bovine cartilage. On the other hand, Gendron et al (32) showed that the N-terminal portion of TIMP-1 at 1 μM failed to inhibit aggrecanase activity in either IL-1α–treated bovine nasal cartilage or retinoic acid–treated porcine articular cartilage. We also tested 0.5 μM TIMP-1 and the N-terminal portion of TIMP-1 (which inhibited IL-1α–induced aggrecanase activity in human cartilage) in IL-1α–treated porcine metacarpophalangeal cartilage, and we found that neither inhibited the release of GAG or aggrecan neoepitopes (Sawaji Y, et al: unpublished observations).
One could hypothesize that the inhibitory effect of FGF-2 on IL-1α–induced aggrecan catabolism in human cartilage is due to an increase in TIMP-1 production. However, although FGF-2 alone increased TIMP-1 production 2-fold, this was not seen when IL-1α was present. The expression of TIMP-3, a strong inhibitor of ADAMTS-4 and ADAMTS-5, also appeared not to be affected by FGF-2. Taken together, these results indicate that neither TIMP-1 nor TIMP-3 was likely to account for the inhibition of aggrecan degradation.
How does FGF-2 inhibit aggrecan catabolism in human cartilage? Our quantitative PCR showed that FGF-2 inhibited the IL-1α–mediated induction of both ADAMTS-4 and ADAMTS-5 mRNA expression. However, there was a time lag between the change in mRNA expression of the enzymes and the release of aggrecan fragments. The earliest time at which aggrecan fragments could be detected was 24 hours, while the induction of ADAMTS-4 and ADAMTS-5 mRNA expression by IL-1α occurred much earlier. It is possible that aggrecanase proteins are produced in increased amounts following the increase in their mRNA caused by IL-1α, but that it takes time for the enzymes to be secreted to cleave aggrecan and to reach levels at which neoepitopes are detected in the medium. It is also possible that proteolytic processing, which could alter the activity and matrix-binding affinity of the enzymes, takes time. For example, C-terminal processing of ADAMTS-4 by membrane type 4 MMP is required for the aggrecanase cleavage in IL-1–treated bovine cartilage (48). We were unable to investigate the molecular forms of the proteinases that were produced because of lack of suitable antibodies. The amount of the enzymes was below the level of immunodetection in human cartilage. However, even these very low amounts may be sufficient to cleave aggrecan.
Our conclusion is that FGF-2 is an anticatabolic growth factor in human cartilage that inhibits aggrecanase-dependent aggrecan degradation, but that does not have a general antiinflammatory effect. FGF-2–mediated inhibition of ADAMTS-4 and/or ADAMTS-5 mRNA expression may in part explain the inhibition of IL-1α–induced aggrecanolysis, but posttranscriptional and posttranslational mechanisms cannot be excluded.
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- MATERIALS AND METHODS
- AUTHOR CONTRIBUTIONS
We thank Prof. Bruce Caterson and Clare Hughes of Cardiff University (Cardiff, UK) for the gift of the anti-ARGS monoclonal antibody, and Dr. John Mort of McGill University (Montreal, Quebec, Canada) for the anti-NITEGE antibody. We also thank our colleague, Prof. Hideaki Nagase, for the antiserum to the AGEG neoepitope, for antibodies to human MMPs and TIMP-1, and for recombinant TIMPs and aggrecan substrate as well as for many helpful discussions. We also thank our surgical colleagues, Tim Briggs and Steve Cannon, at Royal National Orthopaedic Hospital (Stanmore and London, UK) for providing specimens.