Articular cartilage is the avascular tissue that forms the articulating surface of all joints. The extracellular matrix (ECM) of this tissue imparts the biomechanical characteristics that are essential for the painless, friction-free gliding of bone against bone during articular movement. A necessary and plentiful component of cartilage ECM is aggrecan, a large proteoglycan consisting of a protein core backbone substituted with many highly sulfated glycosaminoglycans. Aggrecan provides cartilage with the ability to resist compressive forces. While many enzymes have been demonstrated to be capable of cleaving the protein backbone of aggrecan (1), analysis of the degradation products in naturally occurring osteoarthritis (OA) reveals that a major cleavage site is within the interglobular domain between amino acids 373 and 374 (2). ADAMTS-4 (aggrecanase 1) and, subsequently, ADAMTS-5 (aggrecanase 2) have been identified as the known enzymes that are most efficiently capable of cleaving aggrecan at that particular site (3, 4).
ADAMTS-4 is a member of the “disintegrin and metalloproteinase with thrombospondin-like repeat” family of proteins. Evidence for the involvement of ADAMTS-4 in joint disease comes from several reports of increased expression after stimulation of articular tissues with inflammatory cytokines (5–9), as well as in vitro findings indicating that ADAMTS-4 is one of the few enzymes that can efficiently cleave aggrecan at the site that is cleaved in naturally occurring disease (10, 11). The complete characterization of the function of ADAMTS-4 is still unclear, and there is evidence that it may be involved in pathologic or physiologic processes in nonarticular organs. Abbaszade et al (4) demonstrated expression of ADAMTS-4 in many normal human tissues, including heart, brain, lung, and skeletal muscle. Matthew and colleagues (12) showed that ADAMTS-4 is expressed in glioma cells and is capable of cleaving brevican, a brain-specific ECM protein. Sandy et al (13) reported that ADAMTS-4 could cleave versican, a widely distributed proteoglycan. Whether these expression patterns and activities implicate ADAMTS-4 in nonarticular cartilage processes remains to be determined.
The growth plate is another area that undergoes dramatic cartilage remodeling. During the physiologic process of endochondral ossification, cells within the cartilaginous growth plates proliferate and lay down significant ECM, the composition of which is similar to that of articular cartilage. This cartilaginous matrix is then enzymatically removed concurrent with subsequent vascular invasion and replacement with bone. Regulation of the ECM during growth plate remodeling has been linked to several enzymatic processes, primarily involving matrix metalloproteinases (MMPs), and animals with disruption of specific MMPs have exhibited growth plate abnormalities (14). The involvement of ADAMTS-4 in normal growth plate remodeling has not been fully investigated, but has been implicated due to identification of the aggrecanase-specific cleavage site both in growth plates and in secondary centers of ossification (15, 16).
ADAMTS-4 is, in summary, a zinc-dependent metalloproteinase that is expressed in many tissues throughout the body, but its function in these tissues is largely unknown. Aggrecanase activity in cytokine-stimulated articular cartilage and human OA articular cartilage has been identified, with ADAMTS-4 and/or ADAMTS-5 implicated as potential mediators of this important pathologic process. This same activity has been identified in normal growth plates, implying that this protein may exert physiologic as well as pathologic activity in skeletal tissues. Herein we describe the generation of a mouse line in which the genomic DNA coding the catalytic domain of ADAMTS-4 was deleted, creating a murine model with which to study the effect of lack of enzymatic activity of this protein throughout development and during growth, maturation, and aging. To further evaluate the contribution of this enzyme to the process of joint disease, joint instability was surgically induced in these mice, and the rate of progression and severity of OA was compared in wild-type (WT) versus genetically manipulated (knockout [KO]) mice. In addition, the degradative process in the cartilage of these KO mice was characterized by analysis of the aggrecan degradation products inducible in the animals.
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- MATERIALS AND METHODS
There has been excitement following the reporting of a putative “aggrecanase” capable of cleaving articular cartilage aggrecan at the site within the core protein that is cleaved in human OA (3). Discovery of this enzyme, ADAMTS-4, was quickly followed by the identification of a second, closely related, protein also capable of cleaving aggrecan at the same site, ADAMTS-5 (4). The findings of studies undertaken to positively implicate these enzymes as present and culpable in the disease process have been more difficult to interpret, however. While levels of expression of ADAMTS-4 enzyme can be quite low in nonmanipulated, diseased tissue, several studies have shown ADAMTS-4 to be inducible by inflammatory stimuli. Yamanishi et al (9) found that synovial fibroblasts from humans with OA or rheumatoid arthritis constitutively expressed both ADAMTS-4 and ADAMTS-5, but only ADAMTS-4 expression could be increased by cytokine stimulation. Bau and colleagues (5) observed minimal levels of ADAMTS-4 mRNA in human OA chondrocytes, but demonstrated increased expression with cytokine stimulation. Malfait et al (20) presented evidence that both enzymes were expressed in human OA cartilage, but the levels of expression appeared quite low.
Questions regarding the true function of these enzymes have also been raised as a result of evidence of widespread expression patterns in human tissues. In particular, ADAMTS-4 was found in lung, skeletal muscle, heart, and brain (4). Matthew and colleagues explored the substrate specificity of the ADAMTS enzymes and found that ADAMTS-4 was capable of cleaving brevican, a brain-specific ECM protein (12), and Sandy et al (13) demonstrated that ADAMTS-4 was also capable of cleaving versican, a widely distributed ECM constituent.
We attempted to investigate the physiologic function of ADAMTS-4 by generating a mouse line in which the catalytic portion of the ADAMTS-4 gene was deleted. Although ADAMTS-4 has several domains with potential nonenzymatic functions, its primary role has thus far been hypothesized to be extracellular proteoglycan degradation. Mice in which ECM-degrading metalloproteinase genes have been deleted have been described by several authors (14, 21–28). Deletion of the MMP-2, MMP-7, MMP-11, and MMP-12 genes caused no abnormalities in unchallenged mice (21–24). It is important to note, however, that despite the lack of abnormalities in the MMP-2–KO mouse, obvious abnormalities have been found in humans with mutations in the MMP-2 gene, in several Saudi Arabian families with inherited osteolysis (29). Mice with deletion of the MMP-9 gene did exhibit some growth plate pathology (14), and both MMP-14–KO and ADAMTS-1–KO mice had significant abnormalities, implicating these metalloproteinases in several physiologic processes (25, 28).
In none of these cited studies was OA induced in the mice until a recent study by Clements et al (27), in which joint instability was created in MMP-3–KO mice as well as in mice with deletion of other genes implicated in the inflammatory cascade. The progression of OA was accelerated in the MMP-3–KO animals, and the authors speculated that perturbation of the degradative pathways caused disruption of regulatory mechanisms. It is notable that deletion of none of these metalloproteinases resulted in embryonic death.
The ADAMTS-4–KO mice in our study had no apparent developmental dysfunction, and their gross appearance at birth was normal. Our failure to detect any expression of ADAMTS-4 mRNA during murine development, despite analysis of embryos each day throughout development and the use of 2 radiolabeled probes applied concurrently, provides evidence that protein expression, if present, must be at a very low level during development. Extensive histologic evaluation of up to 36 tissues, including joints and the brain, harvested from 14–18-week-old animals and 1-year-old animals, revealed no indication of developmental or growth deficiencies. Apparently, ADAMTS-4 enzymatic activity was not necessary for normal development, growth, or function of any organ examined, although this does not take into consideration potential requirements for such activity in response to physiologic challenge. There are several examples of KO mice that have had normal gross appearance but have responded abnormally to challenge (22, 24, 30, 31).
ADAMTS-4–KO mice can still produce mRNA of the truncated form of the gene, and therefore may potentially secrete protein with no enzymatic function. We were unable to identify ADAMTS-4 protein in tissue extracts from KO or WT mice despite obvious evidence of activity. If the inactive protein were produced in these animals, findings in the KO mice would mimic the result of inhibition of ADAMTS-4 enzymatic activity by therapeutic intervention.
An attempt to identify the mRNA in normal, WT murine femorotibial joints by in situ hybridization techniques revealed that the ADAMTS-4 message was present in the proximal tibial growth plate, but there was a lack of hybridization in the articular cartilage. Aggrecanase activity was confirmed by appearance of the TEGE373 neoepitope in WT, but not KO, mouse growth plates. This implicates ADAMTS-4, and not ADAMTS-5 or any other aggrecanase, as the source of aggrecanase activity in these growth plates. It was therefore surprising that lack of this activity in the KO animals did not result in abnormal skeletal remodeling. At 1 year of age, WT and KO animals were the same size, and sensitive quantification of long bone length did not reveal any differences between WT and KO animals. One possible explanation is that ADAMTS-4–mediated aggrecan degradation during skeletal remodeling is only a minor fraction of the remodeling activity and can easily be compensated for by the activity of other proteinases such as MMPs.
Data generated by others has demonstrated that ADAMTS-4 mRNA and/or activity is inducible in articular tissues following inflammatory stimuli (5–9, 31). It is therefore possible that ADAMTS-4 activity is not required for normal ECM turnover, but is induced in inflammatory diseases such as arthritis. In order to create an inflammatory milieu and precipitate the OA process, we surgically induced joint instability in the mice. OA developed as expected, and the disease progressed over the course of 8 weeks. There was no difference in the rate or severity of disease progression in the ADAMTS-4–KO mice compared with WT mice.
Three hypotheses that could explain the apparent lack of importance of ADAMTS-4 in murine OA are 1) that aggrecanases are not active in the articular cartilage of the mouse, 2) that MMPs take over the degradation of aggrecan if aggrecanases are not active, and 3) that aggrecanases other than ADAMTS-4 are the primary mediators of aggrecan destruction in OA. To test these hypotheses, we removed articular cartilage from the femoral heads of KO and WT mice and cultured them in the presence of inflammatory cytokines. Destruction and release of proteoglycan into the media was equivalent with specimens from WT and KO mice. We then evaluated the site of cleavage of the aggrecan in the cartilage, using immunohistochemical techniques and Western blot analysis of released proteoglycan. Comparison of the TEGE373 neoepitope left within the cartilage matrix demonstrated that this aggrecanase-specific neoepitope was present in both KO and WT mice, with relatively equal staining intensity. We also identified this cleavage product released into the media.
The TEGE373 antibody was used for analysis based on evidence that the G1 fragment of aggrecan with the TEGE373 C-terminus is released from articular cartilage following IL-1–induced aggrecanase induction (32–35). It was apparent from the results that aggrecan was still being cleaved by an aggrecanase in the operated articular cartilage from ADAMTS-4–KO mice. Similar immunostaining of cartilage from the murine OA knees demonstrated TEGE373 neoepitope at disparate and spotty sites within the joint, depending on the stage of the OA process (results not shown). It is notable that the TEGE373 neoepitope appeared throughout the ECM in the cytokine-stimulated cartilage but was largely intracellular in the growth plates and OA tissue. The profound induction of aggrecanase activity by cytokines, and the choice of the optimal time for analysis, resulted in significant TEGE373 remaining within the ECM of the in vitro–cultured cartilage; the TEGE373 would subsequently disappear. The internalization of the G1 domain of aggrecan has been described (36, 37) and could explain the obvious intracellular staining during the more normal processes of growth plate remodeling and OA. It is likely that TEGE373 within the ECM had disappeared from these tissues by the time of analysis.
In summary, the results of the analysis of the ADAMTS-4–KO mice demonstrated that ADAMTS-4 enzymatic activity is not required for normal development, growth, or homeostasis. Because we do not know whether the nonenzymatic portions of this protein are translated in the KO mice, it is possible that the function of the ADAMTS-4 protein in nonarticular tissues may be through the thrombospondin-like or disintegrin domains. Although there was evidence of significant ADAMTS-4–mediated aggrecan degradation in growth plates, deletion of this gene did not affect skeletal growth or remodeling. When the joints of the ADAMTS-4–KO mice were subjected to mechanical instability to induce OA, there was no reduction in the rate or severity of aggrecan loss or disease progression. Challenge of cartilage from these mice with inflammatory cytokines also initiated aggrecan cleavage, apparently by a non–ADAMTS-4 “aggrecanase”; the site of cleavage of the aggrecan core protein was the site designated the “aggrecanase” cleavage site, TEGE373–374ARGS.
Based on the results of the current study, it is apparent that ADAMTS-4 is not the aggrecanase responsible for aggrecan degradation in murine osteoarthritis. Whether this observation extends to human OA is yet to be determined; it is possible that expression and activity of ADAMTS-4 in the mouse joint differs from that in other species. This is certainly the case with other matrix-degrading enzymes, most notably MMP-1 (38, 39). ADAMTS-5 has clearly been implicated as a potent aggrecanase (4). Others have also reported that ADAMTS-1 can cleave aggrecan at the TEGE373–374ARGS cleavage site, albeit with greatly reduced efficiency (40, 41). ADAMTS-9 has also been shown to be capable of inefficient cleavage of aggrecan at a putative “aggrecanase” site (42). Elucidation of which gene encodes the enzyme responsible for human degradative diseases of articular cartilage will undoubtedly require examination of human diseased tissue.