The structural integrity of articular cartilage is determined principally by homeostasis of the 2 major macromolecules of the extracellular matrix, type II collagen and the chondroitin sulfate–rich proteoglycan aggrecan. In healthy tissue, there is a balance between anabolic (synthetic) and catabolic (degradative) processes that allows matrix turnover. Excessive catabolic activity results in matrix breakdown, a hallmark of osteoarthritis (OA). One key early event in matrix breakdown is loss of aggrecan, which is caused by aggrecanase enzymes, members of the ADAMTS family. In humans, ADAMTS-4 and ADAMTS-5 are thought to be the major aggrecanases in cartilage (1, 2). In the mouse, deletion of ADAMTS-5, but not ADAMTS-4, was shown to protect against the development of OA and inflammatory arthritis, suggesting that ADAMTS-5 is the main murine aggrecanase (3, 4).
We have identified fibroblast growth factor 2 (FGF-2) as a potential regulatory molecule in articular cartilage. It is bound to the heparan sulfate chains of the proteoglycan perlecan in the pericellular matrix of human and porcine cartilage, where it acts as a mechanotransducer (5, 6). Upon loading, FGF-2 is made available to cell surface tyrosine kinase receptors and activates intracellular signaling pathways including ERK, one of the MAPKs (7). FGF-2 is also released from the pericellular pool upon physical injury to the tissue (8).
In order to determine the role of FGF-2 in articular cartilage, we recently investigated the influence of FGF-2 on the breakdown of aggrecan in human articular cartilage explants. We found that FGF-2 suppressed interleukin-1 (IL-1)– or tumor necrosis factor–stimulated aggrecanase activity in explants of normal knee cartilage in a dose-dependent manner (9). Because of these findings, we investigated whether FGF-2 was chondroprotective in vivo. Fgf2−/− mice are viable, fertile, and morphologically indistinguishable from their wild-type (WT) littermates under normal conditions (10). We examined the knee articular cartilage of naive Fgf2−/− and Fgf2+/+ mice, and then we compared both age-related cartilage degeneration in Fgf2−/− and Fgf2+/+ mice and cartilage degeneration following surgical destabilization of the medial meniscus (DMM), a well-established model of OA.
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This is the first description of an endogenous matrix-bound growth factor acting as a chondroprotective agent in vivo; mice deficient in FGF-2 developed accelerated OA with age or following surgical destabilization of the knee. Our earlier observation that FGF-2 inhibits IL-1–driven aggrecanolysis in human explants (9) suggests that the chondroprotective effect of FGF-2 might be due, at least in part, to suppression of aggrecanolysis mediated by ADAMTS-5. Indeed, Adamts5 mRNA was superinduced in Fgf2−/− mice compared with Fgf2+/+ mice 2 weeks following DMM surgery. We were unable to visualize ADAMTS-5 protein due to lack of suitable antibodies and the likely low abundance of the enzyme, although we were able to demonstrate ADAMTS-mediated aggrecanolysis in the cartilage of both Fgf2−/− and Fgf2+/+ mice.
Regulation of ADAMTS-5 gene expression has not been extensively studied. It was initially described as a constitutively expressed aggrecanase in bovine (16, 17) and human (18) chondrocytes, although we and others have demonstrated regulation by inflammatory cytokines in murine (4), bovine (19), and human (9) tissue. The putative promoter region of the gene has predicted binding sites for a number of transcription factors, including NF-κB, which are likely to be involved in gene expression following activation of inflammatory signaling (20). It is not clear how FGF-2 influences ADAMTS-5 gene expression in cytokine-treated cartilage explants. The mechanism by which this occurs in vivo is likely to be yet more complicated because of the expression and effects of FGF-2 in tissues other than articular cartilage.
It is possible that FGF-2 was in part inhibiting aggrecanase activity through induction of TIMP-3, one of the 4 TIMP family members, which is a high-affinity inhibitor of ADAMTS-4 and ADAMTS-5 (21). However, we found no evidence that FGF-2 regulated the expression of TIMP-3 mRNA. Indeed, Timp3 mRNA was not differentially expressed in Fgf2−/− and Fgf2+/+ mice following DMM surgery or in vitro following stimulation of human cartilage with exogenous FGF-2 or IL-1 (9).
It is not known what signals and which cells are responsible for aggrecanase expression following surgical destabilization of the knee. Although there is little or no synovitis seen in this model when cartilage degeneration is occurring (after 4 weeks), it is possible that cytokine production within the synovium or joint cavity drives proteinase production. It is also possible that proteinase expression is directly induced in the chondrocyte by altered joint biomechanics. Destabilization of the meniscus is likely to have 2 main consequences following the initial cutting injury. The first is increased load transmitted through the weight-bearing region of the joint. The second is joint instability resulting in increased shear stress over the articular surfaces. It is not known whether chondrocytes are able to sense and respond to such changes, but our previous finding that simple cutting of cartilage is sufficient to activate JNK and p38 MAPK pathways and to induce inflammatory response genes such as IL-1 in the chondrocyte (22) lends support to such a theory.
The role of FGF-2 in cartilage has been most studied in the growth plate, where it has an inhibitory effect on chondrocyte proliferation. Gain-of-function mutations in human FGF receptor 3 (FGFR-3) (23–27) and overexpression of FGF-2 (28) or intravenous treatment of mice with recombinant FGF-2 (29) all result in a reduction in the proliferating zone of the growth plate and in subsequent shortening of the long bones (achondroplastic dwarfism). The absence of a developmental phenotype in Fgf2−/− mice is thought to be due to compensation by other FGF family members such as FGF-18 which are present in the growth plate (30). It is possible that Fgf2−/− mice exhibited a mild subclinical chondrodysplasia that rendered the cartilage more susceptible to OA in adult life. However, subcutaneous delivery of FGF-2 was able to slow arthritis in Fgf2−/− mice to levels seen in Fgf2+/+ animals, suggesting that accelerated OA in Fgf2−/− mice was not due to an intrinsic matrix weakness that had arisen during development, but rather that it was due to the loss of FGF-2–mediated suppression of matrix breakdown in postnatal tissue.
The disparate functions of FGF-2 in pre- and postnatal cartilage may be due to differences in receptor expression; FGFR-3 expression is high in the growth plate (31), but FGFR-1 and FGFR-2 are the predominant receptors in healthy mature articular cartilage (Vincent T, Chia S-L: unpublished observations), and it is probably through these that the anticatabolic effects of FGF-2 are occurring. Our results suggest that other FGFs do not compensate for the loss of FGF-2 in adult cartilage. This may be because of the high relative abundance of FGF-2 in articular cartilage compared with other FGFs, and also because of the mechanical control of its bioavailability, which ensures that FGF-2–mediated signaling occurs during weight bearing as well as following chondral injury (7, 8).
The role of FGF-2 in tissue injury responses in other organs has also emerged in recent years; Fgf2−/− mice have delayed healing following excisional skin wounding (32), and FGF-2 up-regulates neurogenesis and protects neurons from degeneration in the adult hippocampus after traumatic brain injury (33). These observations identify FGF-2 as a tissue remodeling cytokine and highlight its importance in tissue homeostasis and repair.
Finally, could loss of the chondroprotective role of FGF-2 contribute to the development of human OA? We have observed strong pericellular staining for perlecan and FGF-2 in OA cartilage, suggesting that their expression is maintained in disease. It is not clear at present whether mechanical signals are unable to activate FGF-2–mediated chondroprotection in damaged tissue or whether anticatabolic processes are simply overwhelmed by degradative ones in established OA. Nonetheless, understanding the molecular basis for these observations should enable us to harness the properties of such anticatabolic cytokines in the future management of OA.
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All authors were involved in drafting the article or revising it critically for important intellectual content, and all authors approved the final version to be published. Dr. Vincent had full access to all of the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis.
Study conception and design. Chia, Sawaji, Inglis, Saklatvala, Vincent.
Acquisition of data. Chia, Burleigh, McLean, Inglis.
Analysis and interpretation of data. Chia, Sawaji, Saklatvala, Vincent.