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.     

Selective enhancement of collagenase-mediated cleavage of resident type II collagen in cultured osteoarthritic cartilage and arrest with a synthetic inhibitor that spares collagenase 1 (matrix metalloproteinase 1)

OBJECTIVE: To examine whether type II collagen cleavage by collagenase and loss of proteoglycan are excessive in human osteoarthritic (OA) articular cartilage compared with nonarthritic articular cartilage, and whether this can be inhibited by a selective synthetic inhibitor that spares collagenase 1 (matrix metalloproteinase 1 [MMP-1]). METHODS: Articular cartilage samples were obtained during surgery from 11 patients with OA and at autopsy from 5 adults without arthritis. The articular cartilage samples were cultured in serum-free medium. A collagenase-generated neoepitope, which reflects cleavage of type II collagen, and proteoglycan glycosaminoglycan (GAG), which predominantly reflects aggrecan release, were assayed in culture media. In addition, cultures were performed using either of 2 synthetic MMP inhibitors, both of which inhibited collagenase 2 (MMP-8) and collagenase 3 (MMP-13), but one of which spared collagenase 1. Cultures were also biolabeled with 3H-proline in the presence and absence of these inhibitors to measure collagen synthesis (as tritiated hydroxyproline) and incorporation in articular cartilage. RESULTS: As a group, cleavage of type II collagen by collagenase was significantly increased in OA cartilage samples. In contrast, proteoglycan (GAG) release was not increased. This release of a collagenase-generated epitope was inhibited by both MMP inhibitors in 2 of 5 nonarthritic samples and in 9 of 11 OA cartilage samples. The inhibitor that spared collagenase 1 was generally more effective and inhibited release from 4 of 5 nonarthritic cartilage samples and the same OA cartilage samples. Group analyses revealed that the inhibition of collagenase neoepitope release by both inhibitors was significant in the OA patient cartilage, but not in the nonarthritic cartilage. Proteoglycan loss was unaffected by either inhibitor. Newly synthesized collagen (predominantly, type II) exhibited increased incorporation in OA cartilage, but only in the presence of the inhibitor that arrested collagenase 1 activity. CONCLUSION: These results further indicate that the digestion of type II collagen by collagenase is selectively increased in OA cartilage, and that this can be inhibited in the majority of cases by a synthetic inhibitor that can inhibit collagenases 2 and 3, but not collagenase 1. The results also suggest that in OA, newly synthesized collagen is digested, but in a different manner than that of resident molecules. Proteoglycan release was not increased in OA cartilage and was unaffected by these inhibitors. Inhibitors of this kind may be of value in preventing damage to type II collagen in human arthritic articular cartilage.     

The pathobiology of focal lesion development in aging human articular cartilage and molecular matrix changes characteristic of osteoarthritis

OBJECTIVE: To determine if early focal lesions seen in aging exhibit molecular changes in the extracellular matrix that are similar to those seen in osteoarthritis (OA) and to examine the interrelationships between matrix degradation and synthesis and how they relate to cartilage turnover. METHODS: Condylar cartilage was obtained postmortem from lesion-free joints and from the lesion (where present as well as) from areas adjacent to and remote from the lesion of 31 knees without signs of joint injury (damage to ligaments or menisci). Cartilage was graded histologically and assayed for type II collagen and proteoglycan aggrecan glycosaminoglycan (GAG) contents and turnover (specifically, type II collagen denaturation and its cleavage by collagenase), type II collagen synthesis (C-propeptide [CPII] content), and aggrecan turnover (846 epitope content). To study the degradation of aggrecan reflected by the release of GAG, we cultured cartilage samples from 10 knees. RESULTS: The more degenerated cartilage from the lesion and adjacent area exhibited significantly more collagen cleavage by collagenase than did cartilage remote from the lesion. Type II collagen denaturation and synthesis were also significantly elevated in the lesion and adjacent cartilage, but neither cleavage nor denaturation correlated with synthesis. Type II collagen content decreased with increasing degeneration, with the lowest levels present in the lesion. Collagen content was indirectly related to denaturation and cleavage adjacent to and remote from the lesion and to denaturation within the lesion. Collagen cleavage and denaturation adjacent to and remote from the lesion were directly interrelated. Cartilage from the lesion contained significantly less GAG than did cartilage adjacent to and remote from the lesion. Aggrecan turnover (846 epitope) was also elevated in both the lesion and adjacent cartilage, whereas GAG release was elevated only in the lesion. GAG and 846 epitope contents were interrelated only at sites remote from the lesion. There was also a direct correlation between collagen and GAG contents in the lesion and in adjacent sites. This correlation was also seen between collagen synthesis (CPII) and the 846 epitope. CONCLUSION: These results demonstrate that lesions seen in aging exhibit molecular changes in matrix turnover similar to those seen in OA articular cartilage at arthroplasty, but not in healthy normal aging cartilage. The direct relationships between type II collagen cleavage and denaturation and the inverse relationship between type II collagen content and cleavage or denaturation implicate collagenase activity and damage to collagen in this loss of collagen during lesion development. The lack of correlation of the increased synthesis with the degradation or content of type II collagen indicates that these aspects of turnover are not coordinated in the pathologic state. However, the direct relationship between collagen and GAG contents in and adjacent to the lesion illustrates the structural interrelationships of collagen and proteoglycan aggrecan molecules. These results suggest that these focal lesions represent the development of early OA and that this involves the progressive damage to articular cartilage surrounding the lesion as part of the process of the development of idiopathic OA.     

Anabolic and catabolic gene expression pattern analysis in normal versus osteoarthritic cartilage using complementary DNA-array technology.

OBJECTIVE: To understand changes in gene expression levels that occur during osteoarthritic (OA) cartilage degeneration, using complementary DNA (cDNA)-array technology. METHODS: Nine normal, 6 early degenerated, and 6 late-stage OA cartilage samples of human knee joints were analyzed using the Human Cancer 1.2 cDNA array and TaqMan analysis. RESULTS: In addition to a large variability of expression levels between different patients, significant expression patterns were detectable for many genes. Cartilage types II and VI collagen were strongly expressed in late-stage specimens, reflecting the high matrix-remodeling activity of advanced OA cartilage. The increase in fibronectin expression in early degeneration suggests that fibronectin is a crucial regulator of matrix turnover activity of chondrocytes during early disease development. Of the matrix metalloproteinases (MMPs), MMP-3 appeared to be strongly expressed in normal and early degenerative cartilage and down-regulated in the late stages of disease. This indicates that other degradation pathways might be more important in late stages of cartilage degeneration, involving other enzymes, such as MMP-2 and MMP-11, both of which were up-regulated in late-stage disease. MMP-11 was up-regulated in OA chondrocytes and, interestingly, also in the early-stage samples. Neither MMP-1 nor MMP-8 was detectable, and MMP-13 and MMP-2 were significantly detectable only in late-stage specimens, suggesting that early stages are characterized more by degradation of other matrix components, such as aggrecan and other noncollagenous molecules, than by degradation of type II collagen fibers. CONCLUSION: This investigation allowed us to identify gene expression profiles of the disease process and to get new insights into disease mechanisms, for example, to develop a picture of matrix proteinases that are differentially involved in different phases of the disease process.     

Characterization of metalloprotease cleavage products of human articular cartilage

OBJECTIVE: To identify, characterize, and compare proteolysis peptide products generated by metalloprotease digests of human articular cartilage. METHODS: Human articular cartilage was digested by the addition of exogenous metalloproteases, including matrix metalloproteinases 2, 3, 8, 9, 12, and 13 and aggrecanases ADAMTS-4 and ADAMTS-5. Proteolyzed peptide products were identified by proteomics methods using mass spectrometry. RESULTS: Complete sequences of the peptides proteolyzed from human articular cartilage, including N- and C-termini and hydroxylated posttranslational modifications, were determined. A wide variety of peptides, originating from types I, II, and III collagen, biglycan, prolargin, fibromodulin, fibronectin, decorin, cartilage oligomeric matrix protein, cartilage intermediate-layer protein, megakaryocyte-stimulating factor, mimecan, aggrecan, and lumican, was analyzed following metalloprotease digestion. Release of peptides varied as a function of time, enzyme specificity, and abundance. Specific type II collagen peptide biomarkers, including those containing the three-quarter-length fragment cleavage site and those containing the domains for helical peptide of type II collagen and C-telopeptide of type II collagen, were observed after release by selected proteases. CONCLUSION: The use of intact cartilage instead of purified protein substrates in the assay allowed for the identification of novel potential substrates and cleavage sites for individual enzymes under more physiologically relevant conditions. Characterization of these cartilage matrix peptides may help in the development of pharmacodynamic biomarkers of cartilage degradation, and also may contribute to an understanding of the bioactive peptides important in chondrocyte signaling.     

Turnover of type II collagen and aggrecan in cartilage matrix at the onset of inflammatory arthritis in humans: relationship to mediators of systemic and local inflammation

OBJECTIVE: To determine in vivo the extent of damage to, and changes in turnover of, articular cartilage type II collagen (CII) and the proteoglycan aggrecan following the onset of inflammatory arthritis in humans, and to examine the hypothesis that there are direct relationships between cartilage biomarkers of damage/turnover and clinical, histologic, and molecular markers of inflammation. METHODS: Synovial fluid (SF) and synovial membrane (SM) were obtained by arthroscopy, and a synovitis score was determined, in 32 patients with rheumatoid arthritis (RA) (13 with early untreated disease, 19 with established disease), 18 with psoriatic arthritis (PsA), and 10 with osteoarthritis (OA). Systemic disease activity markers were recorded, and SM CD3+ T cells, CD4+ T cells, CD68+ macrophages, and lining layer hyperplasia were quantified. SF levels of tumor necrosis factor alpha (TNFalpha), interleukin-10 (IL-10), matrix metalloproteinase 1 (MMP-1), MMP-3, Col2-3/4C(Long mono) neoepitope (C2C) (reflecting collagenase cleavage of cartilage CII), C-propeptide of type II procollagen (PIICP) (a biosynthesis marker), keratan sulfate (KS), and the 846 epitope of aggrecan (turnover) were measured by enzyme-linked immunosorbent assay or radioimmunoassay. RESULTS: Levels of cartilage degradation products in early RA or early PsA were not elevated above levels in OA, although in early inflammatory arthritis, TNFalpha and MMP-1 levels were similar to those observed in late inflammatory disease and higher than those in OA. PIICP was reduced in early RA. Correlations were observed between the SF C2C neoepitope level and the Health Assessment Questionnaire score, C-reactive protein level, plasma viscosity, synovitis score, and SF TNFalpha and MMP-1 levels. KS epitope content was reduced in direct relation to SM macrophage infiltration in the sublining and lining layers and in the presence of elevated SF MMP-3. Both SF MMP-1 and SF MMP-3 levels correlated with CD4+ T cell infiltration and lining layer hyperplasia in the SM, and MMP-1 levels correlated with lining layer CD68 levels, but TNFalpha and IL-10 levels did not. CONCLUSION: Except for CII synthesis, there were no significant changes in extracellular matrix turnover of aggrecan or CII in the early stages of human inflammatory arthritis. However, the direct correlation between the increases in TNFalpha and MMP-1 production and collagen degradation suggests that collagenase cleavage of cartilage collagen is related to the activities of TNFalpha and MMP-1. The reduction in CII synthesis in early RA may contribute to the developing pathology, since a lack of synthesis of this molecule would inhibit maintenance of cartilage matrix.     

Evidence of a novel aggrecan-degrading activity in cartilage: Studies of mice deficient in both ADAMTS-4 and ADAMTS-5

OBJECTIVE: To characterize aggrecan catabolism and the overall phenotype in mice deficient in both ADAMTS-4 and ADAMTS-5 (TS-4/TS-5 Delta-cat) activity. METHODS: Femoral head cartilage from the joints of TS-4/TS-5 Delta-cat mice and wild-type mice were cultured in vitro, and aggrecan catabolism was stimulated with either interleukin-1alpha (IL-1alpha) or retinoic acid. Total aggrecan release was measured, and aggrecanase activity was examined by Western blotting using neoepitope antibodies for detecting cleavage at EGE 373-374 ALG, SELE 1279-1280 GRG, FREEE 1467-1468 GLG, and AQE 1572-1573 AGEG. Aggrecan catabolism in vivo was examined by Western blotting of cartilage that had been extracted immediately ex vivo. RESULTS: TS-4/TS-5 Delta-cat mice were viable, fertile, and phenotypically normal. TS-4/TS-5 Delta-cat cartilage explants did not release aggrecan in response to IL-1alpha, and there was no detectable increase in aggrecanase neoepitopes. TS-4/TS-5 Delta-cat cartilage explants released aggrecan in response to retinoic acid. There was no retinoic acid-stimulated cleavage at either EGE 373-374 ALG or AQE 1572-1573 AGEG. There was a low level of cleavage at SELE 1279-1280 GRG and major cleavage at FREEE 1467-1468 GLG. Ex vivo, cleavage at FREEE 1467-1468 GLG was substantially reduced, but still present, in TS-4/TS-5 Delta-cat mouse cartilage compared with wild-type mouse cartilage. CONCLUSION: An aggrecanase other than ADAMTS-4 and ADAMTS-5 is expressed in mouse cartilage and is up-regulated by retinoic acid but not IL-1alpha. The novel aggrecanase appears to have different substrate specificity from either ADAMTS-4 or ADAMTS-5, cleaving E-G bonds but not E-A bonds. Neither ADAMTS-4 nor ADAMTS-5 is required for normal skeletal development or aggrecan turnover in cartilage.     

A fibronectin fragment induces type II collagen degradation by collagenase through an interleukin-1-mediated pathway

OBJECTIVE: To examine the effects of a fibronectin (FN) fragment containing the COOH-terminal heparin-binding domain (HBFN-f) on chondrocyte-mediated type II collagen (CII) cleavage by collagenase and proteoglycan (PG) degradation in articular cartilage in explant culture. METHODS: Intact FN or HBFN-f was added to explant cultures of mature bovine articular cartilage. We investigated collagenase-mediated cleavage of CII caused by HBFN-f in explant cultures using a new immunoassay for detection and measurement of the primary collagenase cleavage site of CII. CII denaturation in cartilage was also measured using a specific enzyme-linked immunosorbent assay. Degradation of PG (principally aggrecan) was analyzed by a dye-binding assay. APMA and/or a matrix metalloproteinase 13 (MMP-13) preferential inhibitor or interleukin-1 receptor antagonist (IL-1Ra) were added to some cultures to examine the presence of latent procollagenase or the involvement of MMP-13 or IL-1, respectively, in cartilage breakdown induced by HBFN-f. Secretion of MMP-3 and MMP-13 into media was detected by immunoblotting. RESULTS: In contrast to intact FN, HBFN-f was shown to stimulate CII cleavage by collagenase in a dose-dependent manner following PG degradation, similar to cartilage breakdown induced by IL-1. Treatment with HBFN-f also resulted in elevated denaturation of CII. Immunoblotting demonstrated that HBFN-f enhanced pro-matrix metalloproteinase 13 (proMMP-13) production as well as that of proMMP-3. APMA, which activates latent proMMPs, enhanced the HBFN-f-mediated cleavage of CII by collagenase. An MMP-13 preferential inhibitor or IL-1Ra suppressed HBFN-f-induced collagen cleavage to control levels. CONCLUSION: Our data demonstrate that HBFN-f can induce early PG degradation and subsequent CII cleavage. The latter is probably mediated by early proMMP-13 induction involving an IL-1-dependent pathway. Activation of latent collagenase is delayed. This new information, together with existing data on other FN fragments, reveals that increased levels of these fragments, found in diseased joints such as in osteoarthritis and rheumatoid arthritis, may stimulate cartilage breakdown by mechanisms of the kind demonstrated in the present study.     

Extracellular sulfatases support cartilage homeostasis by regulating BMP and FGF signaling pathways

The balance between anabolic and catabolic signaling pathways is critical in maintaining cartilage homeostasis and its disturbance contributes to joint diseases such as osteoarthritis (OA). A unique mechanism that modulates the activity of cell signaling pathways is controlled by extracellular heparan endosulfatases Sulf-1 and Sulf-2 (Sulfs) that are overexpressed in OA cartilage. This study addressed the role of Sulfs in cartilage homeostasis and in regulating bone morphogenetic protein (BMP)/Smad and fibroblast growth factor (FGF)/Erk signaling in articular cartilage. Spontaneous cartilage degeneration and surgically induced OA were significantly more severe in Sulf-1^−/− and Sulf-2^−/− mice compared with wild-type mice. MMP-13, ADAMTS-5, and the BMP antagonist noggin were elevated whereas col2a1 and aggrecan were reduced in cartilage and chondrocytes from Sulf^−/− mice. Articular cartilage and cultured chondrocytes from Sulf^−/− mice showed reduced Smad1 protein expression and Smad1/5 phosphorylation, whereas Erk1/2 phosphorylation was increased. In human chondrocytes, Sulfs siRNA reduced Smad phosphorylation but enhanced FGF-2-induced Erk1/2 signaling. These findings suggest that Sulfs simultaneously enhance BMP but inhibit FGF signaling in chondrocytes and maintain cartilage homeostasis. Approaches to correct abnormal Sulf expression have the potential to protect against cartilage degradation and promote cartilage repair in OA.     

Differences in type II collagen degradation between peripheral and central cartilage of rat stifle joints after cranial cruciate ligament transection

OBJECTIVE: Type II collagen degradation is thought to be the key process in cartilage degradation during the development of osteoarthritis (OA). In this study, we investigated the kinetics of type II collagen degradation during surgically induced OA. METHODS: Experimental OA was induced in male Wistar rats by transecting the cranial (anterior) cruciate ligament (CCL). Hematoxylin and eosin staining was used to study overall cartilage degradation, while immunostained sections were used to demonstrate denatured type II collagen (Col2-3/4m antibody) and the collagenase cleavage site in type II collagen (Col2-3/ 4Cshort antibody). RESULTS: During the first 3-4 weeks, cartilage destruction, associated with chondrocyte death, proteoglycan depletion, and a marked increase in the collagenase cleavage neoepitope, was mainly located at the margins of the cartilage. From weeks 3-4, the central part of the cartilage showed increased surface fibrillation and apparent chondrocyte death. In these areas, increased denatured type II collagen staining but little cleavage-site staining was present. CONCLUSION: These results indicate that cartilage degradation after CCL transection in the rat consists of 2 phases. An early phase located at the cartilage margins and a late phase located at the central part of the cartilage. In the early phase, collagenase-dependent cartilage damage occurred. During the late phase, the level of type II collagen denaturation increased.     

Adenoviral-mediated transfer of TGF-beta1 but not IGF-1 induces chondrogenic differentiation of human mesenchymal stem cells in pellet cultures

OBJECTIVE: The objective of the present study was to investigate the potential of application of growth factor genes to induce chondrogenic differentiation of human-derived mesenchymal stem cells (MSCs). The growth factor genes evaluated in the present study were transforming growth factor 1 (TGF-beta1) and insulin-like growth factor 1 (IGF-1). METHODS: Human MSCs were transduced with the adenoviral vectors carrying either TGF-beta1 or IGF-1 (AdTGF-beta1 and AdIGF-1 respectively) or a combination of both growth factor genes at different multiplicities of infection (MOI) and were then made into pellets. Pellets were also made from nontransduced cells and maintained in culture medium supplemented with 10 ng/mL of TGF-beta1. At specified time points, histological analysis, cartilage matrix gene expression, and immunofluorescence were performed to determine the extent of chondrogenic differentiation. RESULTS: MSCs transduced with the AdTGF-beta1 demonstrated robust chondrogenic differentiation, while those made from AdIGF-1 did not. AdTGF-beta1 pellets demonstrated aggrecan gene expression as early as day 3 of pellet culture, while type II collagen gene expression was detected by day 10 of culture. The AdIGF-1, alone or in combination with TGF-beta1 pellets, did not show any type II collagen gene expression at any time point. By immunofluoresecence, type X collagen was distributed throughout the matrix in TGF-beta1 protein pellets while the growth factor gene pellets displayed scant staining. CONCLUSION: The results suggest that sustained administration of TGF-beta1 may be more effective in suppressing terminal differentiation than intermittent dosing and thus effective for cartilage repair.     

Differential matrix degradation and turnover in early cartilage lesions of human knee and ankle joints

OBJECTIVE: To determine whether there are differences in matrix turnover within early cartilage lesions of the ankle (talocrural) joint compared with the knee (tibiofemoral) joint that may help explain differences in the prevalence of osteoarthritis in these 2 joints. METHODS: Cartilage removed from lesions of the tali and femoral condyles was analyzed for type IIB collagen messenger RNA, C-terminal type II procollagen propeptide (CPII), the collagenase cleavage neoepitope (Col2-3/4C(short)), and the denaturation epitope (Col2-3/4m). The content of collagen, glycosaminoglycan, and epitope 846 of aggrecan was quantitated. RESULTS: In ankle lesions, there was an up-regulation of markers of synthesis (CPII [P = 0.07]; epitope 846 [P < or = 0.0001]), but these were down-regulated in the knee (CPII [P = 0.1]; epitope 846 [P = 0.004]). In lesions of the knee, but not the ankle, there was an up-regulation of collagen degradation markers (P = 0.008). On a molar basis, there was 24 times more cleavage epitope than denaturation epitope in knee lesions compared with ankle lesions. CONCLUSION: The up-regulation of matrix turnover that is seen in early cartilage lesions of the ankle would appear to represent an attempt to repair the damaged matrix. The increase in collagen synthesis and aggrecan turnover seen in ankle lesions is absent from knee lesions. Instead, there is an increase in type II collagen cleavage. Together with the differences in collagen denaturation, these changes point to an emphasis on matrix assembly during early lesion development in the ankle and to degradation in the knee, resulting in fundamental differences in matrix turnover in these lesions.     

MMP-8 is only a minor gene product of human adult articular chondrocytes of the knee

OBJECTIVE: The initial degradation of collagen fibrils during osteoarthritic cartilage destruction depends on the cleavage at the collagenase site, for which there exist three major candidate enzymes: collagenase 1 (MMP-1), collagenase 2 (MMP-8), and collagense 3 (MMP-13). The objective of this study was to determine the quantitative expression as well as distribution levels in normal and osteoarthritic cartilage and synovium and in cultured articular chondrocytes with and without stimulation by Il-1 beta. METHODS: Conventional and online PCR technology and immunohistochemistry were used to determine MMP-8 expression levels on the mRNA and protein level. RESULTS: Whereas conventional PCR analysis could demonstrate the presence of MMP-8 mRNA in normal and osteoarthritic chondrocytes, online quantitative PCR showed that only very minor amounts of MMP-8 mRNA expression is found in articular chondrocytes in vivo (and in vitro) and that there is no significant upregulation in osteoarthritic cartilage in vivo nor by Il-1 beta in vitro. The in vivo results were confirmed by the absence of significant protein staining with monoclonal antibodies for MMP-8 in normal and osteoarthritic chondrocytes. CONCLUSIONS: The presented results confirm the presence of a very minor MMP-8 expression by articular chondrocytes, but clearly question the hypothesis that MMP-8 is a major cartilage matrix degrading protease and is involved in enhanced cartilage matrix breakdown in osteoarthritic cartilage degeneration or by Il-1 beta stimulation in vitro.     

Thyroid hormone enhances aggrecanase-2/ADAM-TS5 expression and proteoglycan degradation in growth plate cartilage

Effects of thyroid hormone on proteoglycan degradation in various regions of cartilage were investigated. In propylthiouracil-treated rats with hypothyroidism, proteoglycan degradation in epiphyseal cartilage during endochondral ossification was markedly suppressed. However, injections of T(4) reversed this effect of propylthiouracil on proteoglycan degradation. In pig growth plate explants, T(3) also induced breakdown of proteoglycan. T(3) increased the release of aggrecan monomer and core protein from the explants into the medium. Accordingly, the level of aggrecan monomer remaining in the tissue decreased after T(3) treatment, and the monomer lost hyaluronic acid-binding capacity, suggesting that the cleavage site is in the interglobular domain. The aggrecan fragment released from the T(3)-exposed explants underwent cleavage at Glu(373)-Ala(374), the major aggrecanase-cleavage site. The stimulation of proteoglycan degradation by T(3) was less prominent in resting cartilage explants than in growth plate explants and was barely detectable in articular cartilage explants. Using rabbit growth plate chondrocyte cultures, we explored proteases that may be involved in T(3)-induced aggrecan degradation and found that T(3) enhanced the expression of aggrecanase-2/ADAM-TS5 (a disintegrin and a metalloproteinase domain with thrombospondin type I domains) mRNA, whereas we could not detect any enhancement of stromelysin, gelatinase, or collagenase activities or any aggrecanase-1/ADAM-TS4 mRNA expression. We also found that the aggrecanse-2 mRNA level, but not aggrecanase-1, increased at the hypertrophic stage during endochondral ossification. These findings suggest that aggrecanse-2/ADAM-TS5 is involved in aggrecan breakdown during endochondral ossification, and that thyroid hormone stimulates the aggrecan breakdown partly via the enhancement of aggrecanase-2/ADAM-TS5.