Up regulation of cathepsin K expression in articular chondrocytes in a transgenic mouse model for osteoarthritis

Objectives: To study the expression of cysteine proteinases, particularly cathepsin K, and their extracellular inhibitor cystatin C in articular cartilage of transgenic Del1 mice which harbour a short deletion mutation in a type II collagen transgene and are predisposed to early onset osteoarthritis.

Methods: Northern analysis was used to measure mRNA levels of cathepsins B, H, K, L, and S, and cystatin C in total RNA extracted from knee joints of Del1 mice, using their non-transgenic litter mates as controls. Immunohistochemistry and morphometry was used to study the distribution of cathepsin K and cystatin C in the knee joints.

Results: Up regulation of cathepsin K mRNA expression was seen in the knee joints of transgenic Del1 mice at the onset of cartilage degeneration. Cathepsin K was found near sites of matrix destruction in articular chondrocytes, particularly in clusters of proliferating cells, and in calcified cartilaginous matrix. In intact articular cartilage of control animals, cathepsin K was only seen in a small number of chondrocytes. Upon aging, control animals also developed osteoarthritis, which was accompanied by increased cathepsin K expression. Cystatin C was mostly localised in and around chondrocytes located in calcified cartilage, with no obvious association with the onset of cartilage degeneration.

Conclusion: The temporospatial distribution of cathepsin K in osteoarthritic cartilage suggests a role for this enzyme in the pathogenesis of osteoarthritis. Because cathepsin K can digest cartilage matrix components it may contribute to the development of osteoarthritic lesions. These data may provide new clues for the development of treatments aimed at preventing cartilage degeneration.

     

Lifelong voluntary joint loading increases osteoarthritis in mice housing a deletion mutation in type II procollagen gene,and slightly also in non-transgenic mice

Objectives: To investigate the effects of voluntary running on the incidence and severity of osteoarthritis (OA) and associated changes in cartilage matrix and subchondral bone in a transgenic Del1 mouse model for OA.

Methods: Del1 mice and their non-transgenic littermate controls were housed from the age of 5–6 weeks to 15 months in individual cages with running wheels. The running activity of each mouse was monitored for the entire 12 month period. Additional Del1 and control mice were housed in individual cages without running wheels. At the end of the experiment the severity of OA was evaluated by light microscopy, and the articular cartilage matrix changes by digital densitometry and quantitative polarised light microscopy.

Results: Lifelong voluntary running increased the incidence and severity of OA significantly in Del1 mice (transgenic runners), and slightly also in non-transgenic runners. Severe OA changes increased from 39% in transgenic non-runners to 90% in transgenic runners (p=0.006) in lateral tibial condyles, and from 24% to 80% (p=0.013) in lateral femoral condyles, respectively. The proteoglycan content of articular cartilage was reduced in transgenic runners in comparison with transgenic non-runners (p=0.0167), but a similar effect was not seen in non-transgenic runners compared with non-transgenic non-runners. No attributable differences were seen in the collagen network of articular cartilage or in the subchondral bone between any of the groups.

Conclusion: The Del1 mutation has earlier been shown to disturb the assembly of the cartilage collagen network and thereby increase the incidence and severity of OA with age. In this study, voluntary running was shown to increase further cartilage damage in the lateral compartments of the knee. This suggests that articular cartilage in Del1 mice is less resistant to physical loading than in control mice. Despite severe OA lesions in the knee joint at the age of 15 months, Del1 mice continued to run voluntarily 2–3 km every night.

     

Increased collagen and aggrecan degradation with age in the joints of Timp3(-/-) mice

OBJECTIVE: To investigate the in vivo effect of an imbalance between metalloproteinases and their inhibitors, tissue inhibitors of metalloproteinases (TIMPs), in mouse articular cartilage. METHODS: Hind joints of Timp3(-/-) and wild-type mice were examined by routine staining and by immunohistochemical analysis using antibodies specific for type X collagen and for the neoepitopes produced on proteolytic cleavage of aggrecan (... VDIPEN and ... NVTEGE) and type II collagen. The neoepitope generated on cleavage of type II collagen by collagenases was quantitated in sera by enzyme-linked immunosorbent assay. RESULTS: Articular cartilage from Timp3-knockout animals (ages > or =6 months) showed reduced Safranin O staining and an increase in ...VDIPEN content compared with cartilage from heterozygous and wild-type animals. There was also a slight increase in ... NVTEGE content in articular cartilage and menisci of Timp3(-/-) animals. Chondrocytes showed strong pericellular staining for type II collagen cleavage neoepitopes, particularly in the superficial layer, in knockout mice. Also, there was more type X collagen expression in the superficial zone of articular cartilage, especially around clusters of proliferating chondrocytes, in the knockout mice. More type II collagen cleavage product was found in the serum of Timp3(-/-) mice compared with wild-type animals. This increase was significant in 15-month-old animals. CONCLUSION: These results indicate that TIMP-3 deficiency results in mild cartilage degradation similar to changes seen in patients with osteoarthritis, suggesting that an imbalance between metalloproteinases and TIMP-3 may play a pathophysiologic role in the development of this disease.     

Increased expression of discoidin domain receptor 2 is linked to the degree of cartilage damage in human knee joints: a potential role in osteoarthritis pathogenesis

OBJECTIVE: To investigate the relationship between increased discoidin domain receptor 2 (DDR-2) expression and cartilage damage in osteoarthritis (OA). METHODS: Full-thickness cartilage tissue samples from 16 human knee joints were obtained and the grade of cartilage damage was evaluated according to the Mankin scale. Expression of DDR-2, matrix metalloproteinase 13 (MMP-13), and MMP-derived type II collagen fragments was visualized immunohistochemically. Moreover, upon stimulation with either type II collagen or gelatin, levels of DDR-2 and MMP-13 messenger RNA (mRNA) in primary human articular chondrocytes were assessed by real-time polymerase chain reaction. RESULTS: Immunohistochemical analysis showed an increase in DDR-2 expression in human articular cartilage, which was correlated with the degree of tissue damage. In parallel, the extent of MMP-13 and type II collagen breakdown products was elevated as a function of increased DDR-2 expression and cartilage damage. Furthermore, in vitro experiments revealed an up-regulation of both DDR-2 and MMP-13 mRNA in human articular chondrocytes after stimulation with type II collagen. CONCLUSION: Our data indicate that 3 factors, DDR-2 expression, MMP-13 expression, and the degree of cartilage damage, are linked, such that DDR-2 promotes tissue catabolism, and tissue degradation promotes DDR-2 up-regulation and activation. Thus, the perpetuation of DDR-2 expression and activation can be seen as a vicious circle that ultimately leads to cartilage destruction in OA.     

Esculetin inhibits cartilage resorption induced by interleukin 1alpha in combination with oncostatin M

OBJECTIVE: To determine if a new inhibitor, esculetin (EST), can block resorption of cartilage. METHODS: Interleukin 1alpha (IL1alpha, 0.04-5 ng/ml) and oncostatin M (OSM, 0.4-50 ng/ml) were used to stimulate the release of proteoglycan and collagen from bovine nasal cartilage and human articular cartilage in explant culture. Proteoglycan and collagen loss were assessed by dimethylmethylene blue and hydroxyproline assays, respectively. Collagenase levels were measured by assay of bioactivity and by enzyme linked immunosorbent assay (ELISA). The effects of EST on the expression of matrix metalloproteinases (MMPs) and tissue inhibitor of metalloproteinase-1 (TIMP-1) in the transformed human chondrocyte cell line T/C28a4 were assessed by northern blot analysis. TIMP-1 protein levels were assayed by ELISA. The effect of EST on the MMP-1 promoter was assessed using a promoter-luciferase construct in transient transfection studies. RESULTS: EST inhibited proteoglycan and collagen resorption in a dose dependent manner with significant decreases seen at 66 microM and 100 microM EST, respectively. Collagenolytic activity was significantly decreased in bovine nasal cartilage cultures. In human articular cartilage, EST also inhibited IL1alpha + OSM stimulated resorption and decreased MMP-1 levels. TIMP-1 levels were not altered compared with controls. In T/C28a4 chondrocytes the IL1alpha + OSM induced expression of MMP-1, MMP-3, and MMP-13 mRNA was reduced to control levels by 250 microM EST. TIMP-1 mRNA levels were unaffected by EST treatment. All cytokine stimulation of an MMP-1 luciferase-promoter construct was lost in the presence of the inhibitor. CONCLUSION: EST inhibits degradation of bovine nasal cartilage and human articular cartilage stimulated to resorb with IL1alpha + OSM.     

Insulin-like growth factor 1 blocks collagen release and down regulates matrix metalloproteinase-1, -3, -8, and -13 mRNA expression in bovine nasal cartilage stimulated with oncostatin M in combination with interleukin 1alpha

OBJECTIVE: To investigate the effect of insulin-like growth factor 1 (IGF1) on the release of collagen, and the production and expression of matrix metalloproteinases (MMPs) induced by the proinflammatory cytokine interleukin 1alpha (IL1alpha) in combination with oncostatin M (OSM) from bovine nasal cartilage and primary human articular chondrocytes. METHODS: Human articular chondrocytes and bovine nasal cartilage were cultured with and without IGF1 in the presence of IL1alpha or IL1alpha + OSM. The release of collagen was measured by an assay for hydroxyproline. Collagenase activity was determined with the diffuse fibril assay using 3H acetylated collagen. The expression of MMP-1, MMP-3, MMP-8, MMP-13, and tissue inhibitor of metalloproteinase 1 (TIMP-1) mRNA was analysed by northern blot. RESULTS: IGF1 can partially inhibit the release of collagen induced by IL1alpha or IL1alpha + OSM from bovine nasal cartilage. This was accompanied by a reduced secretion and activation of collagenase by bovine nasal cartilage. IGF1 can also down regulate IL1alpha or IL1alpha + OSM induced MMP-1, MMP-3, MMP-8, and MMP-13 mRNA expression in human articular chondrocytes and bovine chondrocytes. It had no significant effect on the production and expression of TIMP-1 mRNA in chondrocytes. CONCLUSION: This study shows for the first time that IGF1 can partially block the release of collagen from cartilage and suggests that down regulation of collagenases by IGF1 may be an important mechanism in preventing cartilage resorption initiated by proinflammatory cytokines.     

Attenuation of osteoarthritis progression by reduction of discoidin domain receptor 2 in mice

Objective

To investigate whether the reduction of discoidin domain receptor 2 (DDR‐2), a cell membrane tyrosine kinase receptor for native type II collagen, attenuates the progression of articular cartilage degeneration in mouse models of osteoarthritis (OA).

Methods

Double‐heterozygous (type XI collagen–deficient [Col11a1^+/−] and Ddr2‐deficient [Ddr2^+/−]) mutant mice were generated. Knee joints of Ddr2^+/− mice were subjected to microsurgical destabilization of the medial meniscus. Conditions of the articular cartilage from the knee joints of the double‐heterozygous mutant and surgically treated mice were examined by histology, evaluated using a modified Mankin scoring system, and characterized by immunohistochemistry.

Results

The rate of progressive degeneration in knee joints was dramatically reduced in the double‐heterozygous mutant mice compared with that in the type XI collagen–deficient mice. The progression in the double‐heterozygous mutant mice was delayed by ∼6 months. Following surgical destabilization of the medial meniscus, the progressive degeneration toward OA was dramatically delayed in the Ddr2^+/− mice compared with that in their wild‐type littermates. The articular cartilage damage present in the knee joints of the mice was directly correlated with the expression profiles of DDR‐2 and matrix metalloproteinase 13.

Conclusion

Reduction of DDR‐2 expression attenuates the articular cartilage degeneration of knee joints induced either by type XI collagen deficiency or by surgical destabilization of the medial meniscus.

     

Increased collagen and aggrecan degradation with age in the joints of Timp3−/− mice

Objective

To investigate the in vivo effect of an imbalance between metalloproteinases and their inhibitors, tissue inhibitors of metalloproteinases (TIMPs), in mouse articular cartilage.

Methods

Hind joints of Timp3^−/− and wild‐type mice were examined by routine staining and by immunohistochemical analysis using antibodies specific for type X collagen and for the neoepitopes produced on proteolytic cleavage of aggrecan (… VDIPEN and … NVTEGE) and type II collagen. The neoepitope generated on cleavage of type II collagen by collagenases was quantitated in sera by enzyme‐linked immunosorbent assay.

Results

Articular cartilage from Timp3‐knockout animals (ages ≥6 months) showed reduced Safranin O staining and an increase in …VDIPEN content compared with cartilage from heterozygous and wild‐type animals. There was also a slight increase in … NVTEGE content in articular cartilage and menisci of Timp3^−/− animals. Chondrocytes showed strong pericellular staining for type II collagen cleavage neoepitopes, particularly in the superficial layer, in knockout mice. Also, there was more type X collagen expression in the superficial zone of articular cartilage, especially around clusters of proliferating chondrocytes, in the knockout mice. More type II collagen cleavage product was found in the serum of Timp3^−/− mice compared with wild‐type animals. This increase was significant in 15‐month‐old animals.

Conclusion

These results indicate that TIMP‐3 deficiency results in mild cartilage degradation similar to changes seen in patients with osteoarthritis, suggesting that an imbalance between metalloproteinases and TIMP‐3 may play a pathophysiologic role in the development of this disease.

     

Osteoarthritis-like changes and decreased mechanical function of articular cartilage in the joints of mice with the chondrodysplasia gene (cho)

OBJECTIVE: To investigate whether heterozygosity for a loss-of-function mutation in the gene encoding the alpha1 chain of type XI collagen (Col11a1) in mice (chondrodysplasia, cho) causes osteoarthritis (OA), and to understand the biochemical and biomechanical effects of this mutation on articular cartilage in knee and temporomandibular (TM) joints. METHODS: Articular cartilage from the knee and TM joints of mice heterozygous for cho (cho/+) and their wild-type littermates (+/+) was examined. The morphologic properties of cartilage were evaluated, and collagen fibrils were examined by transmission electron microscopy. Immunohistochemical staining was performed to examine the protein expression levels of matrix metalloproteinase 3 (MMP-3) and MMP-13 in knee joints. In 6-month-old animals, fixed-charge density was determined using a semiquantitative histochemical method, and tensile stiffness was determined using an osmotic loading technique. RESULTS: The diameter of collagen fibrils in articular cartilage of knee joints from heterozygous cho/+ mice was increased relative to that in control cartilage, and histologic analysis showed OA-like degenerative changes in knee and TM joints, starting at age 3 months. The changes became more severe with aging. At 3 months, protein expression for MMP-3 was increased in knee joints from cho/+ mice. At 6 months, protein expression for MMP-13 was higher in knee joints from cho/+ mice than in joints from their wild-type littermates, and negative fixed-charge density was significantly decreased. Moreover, tensile stiffness in articular cartilage of knee joints from cho/+ mice was moderately reduced and was inversely correlated with the increase in articular cartilage degeneration. CONCLUSION: Heterozygosity for a loss-of-function mutation in Col11a1 results in the development of OA in the knee and TM joints of cho/+ mice. Morphologic and biochemical evidence of OA appears to precede significant mechanical changes, suggesting that the cho mutation leads to OA through a mechanism that does not initially involve mechanical factors.     

Requirement of mitogen-activated protein kinase for collagenase production by the fibronectin fragment in human articular chondrocytes in culture

Abstract

Fibronectin fragments have been shown to up-regulate matrix metalloproteinase production in chondrocytes. We investigated the roles of mitogen-activated protein kinase (MAPK) pathways activated by the COOH-terminal heparin-binding fibronectin fragment (HBFN-f) in collagenase production by human chondrocytes in culture. In articular cartilage explant culture, HBFN-f stimulated type II collagen cleavage by collagenase in association with increased secretion of MMP-1 and MMP-13. In human articular chondrocytes, HBFN-f induced the collagenases with activation of the extracellular signal-regulated kinase (ERK), p38, and the c-Jun NH₂-terminal kinase (JNK). PD98059 that inhibits the ERK pathway blocked HBFN-f-stimulated production of MMP-1 and MMP-13 in explant culture. SB203580 at 1?µM, the concentration that inhibits p38 only, partially suppressed HBFN-f-induced collagenase production, whereas at 10?µM, the inhibitor that blocks both p38 and JNK almost completely inhibited collagenase induction. PD98059 and SB203580 individually blocked HBFN-f-increased cleavage of type II collagen in the explant culture, although 10?µM SB203580 strongly inhibited the collagen cleavage compared with 1?µM of the inhibitor. These results indicate that collagenase production leading to type II collagen cleavage in cartilage explants requires ERK, p38, and JNK.     

Comparison of the degradation of type II collagen and proteoglycan in nasal and articular cartilages induced by interleukin-1 and the selective inhibition of type II collagen cleavage by collagenase

OBJECTIVE: To compare interleukin-1alpha (IL-1alpha)-induced degradation of nasal and articular cartilages in terms of proteoglycan loss and type II collagen cleavage, denaturation, and release; to examine the temporal relationship of these changes; and to investigate the effects of an inhibitor of collagenase 2 and collagenase 3 on these catabolic processes. METHODS: Discs of mature bovine nasal and articular cartilages were cultured with or without human IL-1alpha (5 ng/ml) with or without RS102,481, a selective synthetic inhibitor of collagenase 2 and collagenase 3 (matrix metalloproteinase 8 [MMP-8] and MMP-13, respectively) but not of collagenase 1 (MMP-1). Immunoassays were used to measure collagenase-generated type II collagen cleavage neoepitope (antibody COL2-3/4C(short)) and denaturation (antibody COL2-3/4m), as well as total type II collagen content (antibody COL2-3/4m) in articular cartilage and culture media. A colorimetric assay was used to measure total proteoglycan concentration (principally of aggrecan) as sulfated glycosaminoglycans (sGAG). RESULTS: IL-1alpha initially induced a decrease in tissue proteoglycan content in nasal cartilage. A progressive loss of proteoglycan was noted during culture in articular cartilages, irrespective of the presence of IL-1alpha. In both cartilages, proteoglycan loss was followed by IL-1alpha-induced cleavage of type II collagen by collagenase, which was often reflected by increased denaturation. The inhibitor RS102,481 had no clear effect on the reduction in proteoglycan content (measured by sGAG) and collagen denaturation in either cartilage, but at 10 nM it inhibited the enhanced cleavage of type II collagen, partially in nasal cartilage and completely in articular cartilage. CONCLUSION: IL-1alpha-induced cleavage and denaturation of type II collagen is observed in both hyaline cartilages and is secondary to proteoglycan loss. It probably involves different collagenases, since there is no evidence of a rate-limiting role for collagenase 1 in articular cartilage, unlike the case for nasal cartilage. Inhibitors of this kind may be of value in the treatment of cartilage damage in arthritis. Also, the ability to detect the release of type II collagen collagenase-generated fragments from degraded cartilage offers the potential to monitor cartilage collagen damage and its control in vivo.     

Retinoic acid and oncostatin M combine to promote cartilage degradation via matrix metalloproteinase-13 expression in bovine but not human chondrocytes

OBJECTIVES: Retinoic acid (RetA) and oncostatin M (OSM) have both been shown to mediate potent effects with respect to extracellular matrix integrity. This study assesses the effects of a RetA + OSM combination on cartilage catabolism. METHODS: Animal and human cartilage samples were used to assess the ability of RetA + OSM to promote the release of collagen and proteoglycan fragments, which was determined by measuring glycosaminoglycan and hydroxyproline, respectively. Total collagenolytic and tissue inhibitor of metalloproteinases (TIMP) inhibitory activities were determined by bioassay, whilst gene expression of matrix metalloproteinases (MMPs) and TIMP-1 were determined by northern blotting. Immunohistochemistry was used to assess the presence of MMP-1 and -13 in resorbing cartilage explants. RESULTS: Both agents alone induced proteoglycan release from bovine cartilage, whilst RetA-induced collagen release was variable. Reproducible and synergistic collagenolysis was observed with RetA + OSM, which appeared to be due to MMP-13. Similar collagen release was observed from porcine cartilage. Conversely, no collagen release was seen with human articular cartilage. In primary human chondrocytes, RetA + OSM failed to induce MMP-1 or -13 but caused a significant increase in TIMP-1 expression. CONCLUSIONS: These novel observations show that the combination of RetA + OSM has profound effects on cartilage matrix turnover, but these effects are species-specific. A better understanding of the mechanism by which this combination differentially regulates MMP and TIMP expression in human chondrocytes could provide valuable insight into new therapeutic strategies aimed at the prevention of cartilage destruction.     

Morphology and phenotype expression of types I, II, III, and X collagen and MMP-13 of chondrocytes cultured from articular cartilage of Kashin-Beck Disease

OBJECTIVE: We investigated the characteristics of cell morphology and expression of types I, II, III, and X collagen and matrix metalloproteinase-13 (MMP-13) of chondrocytes from articular cartilage of adult patients with Kashin-Beck Disease (KBD) in vitro to understand the pathogenesis in chondrocytes. METHODS: Samples of articular cartilage were divided into 2 groups: KBD group (8 samples, 8 cases) and the control (8 samples, 8 cases). KBD patients were diagnosed according to "Pathological Criteria to Diagnose KBD in China." Hyaline cartilage was digested with collagenase into cell suspensions and cultured in monolayers. Chondrocyte ultrastructure was observed by electron microscope at 10th day in vitro. Primary articular chondrocytes were seeded on microscope slides and immunostained on 12th day of cultivation for types I, II, III, and X collagens and MMP-13. Positive findings were counted by light microscopy and confirmed by flow cytometric analyses. RESULTS: Considerable amounts of vacuoles and distorted nuclei, as well as thickening and irregular arrangement of collagen fibrils, were seen in the KBD samples by electron microscopy. Types I, III, and X collagen were stained in the KBD, but not in the control cultures. The percentages of positive staining for type II collagen were significantly lower in KBD than those in controls (t col II = -5.54, p < 0.001), and for MMP-13 in the KBD group were significantly higher (t MMP-13 = 3.70, p < 0.01). CONCLUSION:Phenotype expressions of types I, II, III, and X collagen and MMP-13 in chondrocytes cultured in vitro were significantly different between the KBD and control cultures, indicating degenerative and hypertrophic changes in chondrocytes of KBD articular cartilage.     

Chondrocyte‐intrinsic Smad3 represses Runx2‐inducible matrix metalloproteinase 13 expression to maintain articular cartilage and prevent osteoarthritis

Objective

To identify mechanisms by which Smad3 maintains articular cartilage and prevents osteoarthritis.

Methods

A combination of in vivo and in vitro approaches was used to test the hypothesis that Smad3 represses Runx2‐inducible gene expression to prevent articular cartilage degeneration. Col2‐Cre;Smad3^fl/fl mice allowed study of the chondrocyte‐intrinsic role of Smad3 independently of its role in the perichondrium or other tissues. Primary articular cartilage chondrocytes from Smad3^fl/fl mice and ATDC5 chondroprogenitor cells were used to evaluate Smad3 and Runx2 regulation of matrix metalloproteinase 13 (MMP‐13) messenger RNA (mRNA) and protein expression.

Results

Chondrocyte‐specific reduction of Smad3 caused progressive articular cartilage degeneration due to imbalanced cartilage matrix synthesis and degradation. In addition to reduced type II collagen mRNA expression, articular cartilage from Col2‐Cre;Smad3^fl/fl mice was severely deficient in type II collagen and aggrecan protein due to excessive MMP‐13–mediated proteolysis of these key cartilage matrix constituents. Normally, transforming growth factor β (TGFβ) signals through Smad3 to confer a rapid and dynamic repression of Runx2‐inducible MMP‐13 expression. However, we found that in the absence of Smad3, TGFβ signals through p38 and Runx2 to induce MMP‐13 expression.

Conclusion

Our findings elucidate a mechanism by which Smad3 mutations in humans and mice cause cartilage degeneration and osteoarthritis. Specifically, Smad3 maintains the balance between cartilage matrix synthesis and degradation by inducing type II collagen expression and repressing Runx2‐inducible MMP‐13 expression. Selective activation of TGFβ signaling through Smad3, rather than p38, may help to restore the balance between matrix synthesis and proteolysis that is lost in osteoarthritis.

     

Pathogenesis of osteoarthritis-like changes in the joints of mice deficient in type IX collagen

OBJECTIVE: To examine the pathogenetic mechanisms of osteoarthritis (OA)-like changes in Col9a1-/- mice, which are deficient in type IX collagen. METHODS: Knee joints and temporomandibular joints (TMJs) from Col9a1-/- mice and their wild-type (Col9a1+/+) littermates were examined by light microscopy. Immunohistochemical staining was performed to examine the expression of matrix metalloproteinase 3 (MMP-3) and MMP-13, degraded type II collagen, and the discoidin domain receptor 2 (DDR-2) in knee joints. Cartilage mechanics were also evaluated for compressive properties by microindentation testing of the tibial plateau and for tensile properties by osmotic loading of the femoral condyle. RESULTS: Histologic analysis showed age-dependent OA-like changes in the knee and TMJs of Col9a1-/- mice starting at the age of 3 months. At the age of 6 months, enhanced proteoglycan degradation was observed in the articular cartilage of the knee and TMJs of the mutant mice. The expression of MMP-13 and DDR-2 protein and the amount of degraded type II collagen were higher in the knee joints of Col9a1-/- mice than in their wild-type littermates at the age of 6 months. Changes in cartilage mechanics were observed in the femoral and tibial plateaus of Col9a1-/- mice at 6 months, including a decrease in the compressive modulus and uniaxial modulus. At 3 and 6 months of age, tibial cartilage in Col9a1-/- mice was found to be more permeable to fluid flow, with an associated compromise in the fluid pressurization mechanism of load support. All of these changes occurred only at medial sites. CONCLUSION: Lack of type IX collagen in Col9a1-/- mice results in age-dependent OA-like changes in the knee joints and TMJs.     

Cleavage of aggrecan at the Asn341-Phe342 site coincides with the initiation of collagen damage in murine antigen-induced arthritis: a pivotal role for stromelysin 1 in matrix metalloproteinase activity.

OBJECTIVE: The destruction of articular cartilage during arthritis is due to proteolytic cleavage of the extracellular matrix components. This study investigates the kinetic involvement of metalloproteinases (MMPs) in the degradation of the 2 major cartilage components, aggrecan and type II collagen, during murine antigen-induced arthritis (AIA). In addition, the role of stromelysin 1 (SLN-1) induction of MMP-induced neoepitopes was studied. METHODS: VDIPEN neoepitopes in aggrecan and collagenase-induced COL2-3/4C neoepitopes in type II collagen were identified by immunolocalization. Stromelysin 1-deficient knockout (SLN1-KO) mice were used to study SLN-1 involvement. RESULTS: In AIA, the VDIPEN epitopes in aggrecan appeared after initial proteoglycan (PG) depletion. The collagenase-induced type II collagen neoepitopes colocalized with VDIPEN epitopes. Remarkably, cartilage from arthritic SLN1-KO mice showed neither the induction of VDIPEN nor collagen cleavage-site neoepitopes during AIA, suggesting that stromelysin is a pivotal mediator in this process. PG depletion, as measured by the loss of Safranin O staining, was similar in SLN1-KO mice and wild-type strains. Furthermore, in vitro induction of VDIPEN epitopes in aggrecan and COL2-3/4C epitopes in type II collagen, on exposure of cartilage to interleukin-1, could not be accomplished in SLN1-KO mice, whereas intense staining was achieved for both epitopes in cartilage of wild-type strains. CONCLUSION: This study emphasizes that SLN-1 is essential in the induction of MMP-specific aggrecan and collagen cleavage sites during AIA. It suggests that SLN-1 is not a dominant enzyme in PG breakdown, but that it activates procollagenases and is crucial in the initiation of collagen damage.