Accelerated proteolysis of aggrecan and the consequent loss of the glycosaminoglycan (GAG)–bearing region of the molecule from articular cartilage during arthritis is an early and central event in disease pathogenesis. Depletion of aggrecan renders the cartilage less resistant to mechanical compression and may be a prerequisite for subsequent matrix metalloproteinase (MMP)–driven destruction of the collagen network (1). Ultimately, erosion of articular cartilage results in unrecoverable joint dysfunction that may necessitate joint replacement surgery. Two predominant enzyme activities have been implicated in aggrecan proteolysis in arthritis, namely, MMPs and aggrecanases. However, based on the detection of specific aggrecan cleavage products, it seems highly likely that the proteolysis of aggrecan in human joint disease is due to aggrecanase (for review, see refs. 2 and3). Turnover of aggrecan is also implicated in normal physiologic processes, such as development of the primary and secondary ossification centers, and in the growth plates of long bones. As in pathologic articular cartilage, aggrecan fragments resulting from both MMP and aggrecanase activity (G1-PEN and G1-EGE, respectively) have been detected in these growth cartilages (4–6).
A number of aggrecanases, defined by their ability to cleave aggrecan at specific Glu–Xaa peptide bonds, have now been identified as members of the ADAMTS family of proteinases, which includes ADAMTS-1, ADAMTS-4, ADAMTS-5, ADAMTS-8, ADAMTS-9, and ADAMTS-15 (7–13). Although cleavage of aggrecan by aggrecanases has been clearly identified in the growth plate and in normal and arthritic articular cartilage, the identity of the responsible enzymes in normal versus pathologic situations and in the different tissues has yet to be resolved. ADAMTS-1, ADAMTS-4, ADAMTS-5, ADAMTS-9, and ADAMTS-15 are expressed in normal human articular chondrocytes (11, 14–16). There are conflicting data on expression change of these potential aggrecanases in osteoarthritic (OA) compared with normal human cartilage, however, with both increased (15–17) and decreased (11) levels being reported. ADAMTS-1, ADAMTS-4, and ADAMTS-5 proteins have been detected in normal and OA human articular cartilage, although from the few studies available, analysis of change in content with disease is not possible (16–18). There have been no studies of ADAMTS expression or protein in human growth cartilage. Increased expression of ADAMTS-5, but not ADAMTS-4, has been reported in rabbit hypertrophic growth plate chondrocytes and in response to thyroid hormone (19). Furin-activated ADAMTS-4 protein has been colocalized with aggrecanase-cleaved aggrecan in the growth plate adjacent to the secondary ossification center but not in the metaphysis (6). ADAMTS-1 is expressed in embryonic limb buds (20) and is present and up-regulated by parathyroid hormone in metaphyseal and diaphyseal bone (21).
It is evident that the contribution of the different aggrecanases in normal and pathologic aggrecanolysis in articular and growth plate cartilage remains unclear. One way to address this issue is to study mice with a null mutation in one or more of the aggrecanases. Targeted disruption of the ADAMTS-4 gene in mice was recently described. The animals showed no musculoskeletal growth abnormalities, no abrogation of aggrecan catabolism in vitro, and the development of arthritis was not impaired in vivo (22). These results suggest that ADAMTS-4 is not the principal aggrecanase in murine cartilage. ADAMTS-1–knockout (KO) mice have also been described, and the phenotype includes developmental abnormalities in renal, adrenal, and adipose tissue and subfertility in females (23–25). These phenotypic abnormalities are consistent with the expression of ADAMTS-1 in the affected tissues (20, 26–28).
In light of the previous identification of ADAMTS-1 expression and/or protein in normal and pathologic articular cartilage (11, 14–16), limb buds (20), and metaphyseal bone (21), we undertook a more in-depth comparison of musculoskeletal development and normal and arthritic cartilage aggrecanolysis in ADAMTS-1–KO mice. In the present study, we examined the expression of ADAMTS-1 in growth plate chondrocyte maturation in wild-type (WT) mice. Bone and joint morphology and development were studied histologically and immunohistologically in WT versus ADAMTS-1–KO mice. To assess the potential contribution of ADAMTS-1 to cartilage destruction in joint disease, an inflammatory model of arthritis was evaluated. Finally, proteolysis of aggrecan was evaluated in WT and ADAMTS-1–KO cartilage using a novel in vitro model of early cartilage degradation.
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- MATERIALS AND METHODS
Proteolysis at both the N341–F and the E373–A peptide bonds in the interglobular domain of aggrecan has been reported in normal and arthritic articular cartilage (for review, see ref. 2). Cleavage at the latter site through the action of the aggrecanases appears to be the principal event in early pathologic aggrecan loss. In contrast, while both cleavage events have been detected in growth cartilage, the relative importance of these events in bone and cartilage growth and development remains unclear (4–6). Furthermore, there are conflicting data on the expression and localization of aggrecanases both in growth cartilage and normal and diseased articular cartilage. ADAMTS-5, but not ADAMTS-4, is expressed in hypertrophic growth plate chondrocytes in vitro (19), but G1-EGE colocalizes with furin-activated ADAMTS-4 and not ADAMTS-5 protein in growth cartilage in vivo (6). Similarly, both increased and decreased ADAMTS-1, ADAMTS-4, and ADAMTS-5 expression has been reported in human OA cartilage (15–17). Defining the proteinases responsible for aggrecan proteolysis in normal versus pathologic aggrecan turnover has important implications for the treatment of arthritic disease.
This is the first study to demonstrate that ADAMTS-1 is expressed in the chondrocytes of the growth plate and, furthermore, that it is up-regulated with chondrocyte hypertrophy. Attempts to demonstrate ADAMTS-1 protein in these cartilages using immunohistochemistry or Western blotting were unsuccessful (data not shown), reflecting the insensitivity of the methods used and the relatively low abundance of ADAMTS enzymes in cartilage (22). Nevertheless, the increase in ADAMTS-1 mRNA we observed parallels that shown for ADAMTS-5 mRNA in rabbit growth plate chondrocytes undergoing hypertrophic differentiation and maturation in vitro and contrasts with the unchanged and very low expression of ADAMTS-4 in these cells (19).
Increased expression of ADAMTS-1 has been reported in bone and osteoblasts in response to parathyroid hormone (PTH), PTH-related protein (PTHrP), and vitamin D3 (21). The increase in ADAMTS-1 in the hypertrophic chondrocytes may therefore be related to the increased expression of the PTH/PTHrP receptor that occurs in prehypertrophic and early hypertrophic chondrocytes (19, 42), although maximal ADAMTS-1 expression was not evident until late-stage hypertrophy. In addition to its proteolytic activity (7, 43, 44), ADAMTS-1 also has anti-angiogenic properties possibly associated with its ability to sequester vascular endothelial growth factor (26, 45, 46). ADAMTS-1 in the lower growth plate could therefore function to inhibit or delay vascular invasion or promote turnover of aggrecan or other matrix components, both of which could be significant for normal growth plate maturation. However, no apparent effects on chondrocyte maturation or growth plate development, as detected by morphology and type X collagen synthesis, were observed in ADAMTS-1–KO mice. This is similar to the lack of growth plate phenotype in ADAMTS-4–KO mice (22) and could be due to redundancy and compensation by other enzymes, such as ADAMTS-5, which are similarly up-regulated by thyroid hormones in the growth plate (19).
However, it is noteworthy that we did not demonstrate aggrecanase-generated G1-EGE in WT or ADAMTS-1–KO growth plates, even after catabolic stimulation that was able to increase this fragment in articular cartilage (Figure 4B). This may suggest that while ADAMTS enzymes are expressed in growth plate chondrocytes, their proteolytic activity is tightly regulated, and minimal aggrecanase cleavage of aggrecan occurs. Glasson et al (22) did demonstrate G1-EGE in growth plates of WT mice but not ADAMTS-4–KO mice; however, this was predominantly cell-associated rather than matrix staining and the mice were mature (14–18 weeks). Cleavage of aggrecan by aggrecanases in growth plates of young mice during their growth phase has not been demonstrated. Similarly, G1-EGE was localized to the secondary center of ossification but not the metaphyseal border of the primary growth plate in rats (5, 6). The proteolytic target of ADAMTS in the growth plate may be cartilage oligomeric matrix protein or fibromodulin (47, 48) rather than aggrecan. Alternatively, secondary proteolysis by MMPs or cathepsin B in the growth plate (but not articular cartilage) could convert the G1-EGE into G1-PEN.
ADAMTS-1 is expressed in normal and arthritic human articular chondrocytes (11, 14, 16). Although we demonstrated ADAMTS-1 expression in mouse articular chondrocytes, we did not observe any abrogation of aggrecan loss in AIA in ADAMTS-1–KO mice. This is consistent with evidence implicating ADAMTS-4 and ADAMTS-5 as the principal aggrecanases in pathologic conditions and with the finding that they are significantly more active than ADAMTS-1 against aggrecan in solution assays (7, 17, 49, 50). Furthermore, the in vitro release of aggrecan from the cartilage of ADAMTS-1–KO mice was identical to that of WT mice, suggesting that ADAMTS-1 has no significant role in this process. These findings with mouse cartilage are consistent with those of Tortorella et al (51), who showed that the soluble aggrecanase activity in conditioned medium from IL-1–stimulated bovine cartilage was not inhibited using an ADAMTS-1 antibody, but could be reduced by 75% and 15% using antibodies against ADAMTS-4 and ADAMTS-5, respectively. However, the lack of effect of ADAMTS-4 ablation on cartilage aggrecan metabolism or arthritis development (22) suggests that ADAMTS-5 may be the principal enzyme involved in these processes in mice.
In the present study, no compensatory increase in mRNA expression for other aggrecanases was found that would explain the lack of cartilage phenotype in the ADAMTS-1–KO mouse. Despite the lack of effect on GAG release, we did consistently observe less G1-EGE in IL-1–stimulated ADAMTS-1–KO cartilage cultures, suggesting that ADAMTS-1 may contribute to the generation of some of this neoepitope. This finding, in conjunction with a lack of change in GAG release, suggests alternative mechanisms for GAG loss in ADAMTS-1–KO cultures rather than compensation by other aggrecanases. There was no consistent difference in the release of intact G1-bearing aggrecan or the distribution of the C-terminal chondroitin sulfate–bearing catabolites between WT and ADAMTS-1–KO cartilage. Furthermore, the compensatory GAG release or potential loss of EGE epitope could not be explained by increased cleavage to generate PEN.
Culture of cartilage explants from humans and animals in which the mass of tissue is not limiting has been used to investigate proteolysis of aggrecan, collagen, and other matrix proteins. There is a need for a similar in vitro culture system using mouse cartilage to take advantage of the increasing availability of genetically modified mice to elucidate the mechanisms and pathways involved in cartilage catabolism. We have characterized a new and simple model in which cartilage and medium from a single femoral head (∼1 mg of tissue) from a 3–4-week-old mouse will provide sufficient aggrecan and aggrecan fragments for GAG analysis by the DMMB assay as well as 4–5 individual Western blots. Mouse cartilage responded in a similar manner to other species, not only with increased aggrecanase activity, but also with release of G1-bearing aggrecan and link protein in response to RA and IL-1, which is likely associated with increased hyaluronan release or breakdown (33, 52–55).
In contrast to other species, G1-PEN was increased in mouse cartilage, particularly following treatment with RA, due to the presence of growth plate as well as articular cartilage. Dose-response curves (not shown) demonstrated that GAG loss from mouse cartilage was maximal at 10 μM RA, whereas 1 μM elicits maximal GAG release from other species (33, 56–58). Collagen cleavage by collagenases was also increased in RA-treated cultures; however, this was not associated with increased MMP-13 even though RA has been shown to increase MMP-13 expression in rodent chondrocytes (59). This may be associated with insufficient sensitivity of the Western blot detection method in the present study. Alternatively, although MMP-13 appears to be the principal interstitial collagenase in rodents (60), MMP-14, which is also up-regulated by RA (59), may be responsible for the collagen cleavage observed in our explant cultures. Whether this collagenolytic activity occurs in the articular or growth plate cartilage was not determined.
In conclusion, although ADAMTS-1 is expressed in articular and growth cartilage and is able to cleave aggrecan at physiologically relevant sites, our results indicate that it does not play a significant nonredundant role in normal cartilage and bone development and growth. Furthermore, lack of protection against pathologic aggrecan loss in vivo or in vitro in the ADAMTS-1–KO mouse suggests that ADAMTS-1 is not a suitable target for modulating cartilage destruction in arthritis.