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.

Synergistic induction of matrix metalloproteinase 1 by interleukin-1alpha and oncostatin M in human chondrocytes involves signal transducer and activator of transcription and activator protein 1 transcription factors via a novel mechanism.

OBJECTIVE: To investigate the mechanism of interleukin-1alpha (IL-1alpha) and oncostatin M (OSM) synergistic regulation of matrix metalloproteinase 1 (MMP-1) in human chondrocytes. METHODS: Using an immortalized human chondrocyte cell line (T/C28a4), we investigated regulation of the MMP-1 gene. Northern blotting and flow cytometric analysis were used to assess changes in receptor, MMP-1, and c-fos expression. Transient transfections using MMP-1 promoter/luciferase constructs, electrophoretic mobility shift assay, and site-directed mutagenesis were used to investigate MMP-1 promoter activation. RESULTS: We found no alteration in the expression of receptors used by these cytokines after stimulation with IL-1alpha/OSM. Using MMP-1 promoter/luciferase reporter constructs, we found that the proximal (-517/+63) region of the MMP-1 promoter was sufficient to support a synergistic activation. A role for activated signal transducers and activators of transcription (STAT-3) was demonstrated, although no binding of STAT-3 to the MMP-1 promoter was found. However, constitutive binding of activator protein 1 (AP-1) was detected, and changes in c-fos expression could modulate promoter activity. CONCLUSION: Since no changes in receptor expression were observed, receptor modulation cannot account for the IL-1alpha/OSM synergy observed. Instead, the interplay of various intracellular signaling pathways is a more likely explanation. STAT activation is required, but STAT proteins do not interact directly with the MMP-1 promoter. We propose that activated STATs stimulate c-fos expression, and changes in expression of the AP-1 components regulate MMP-1 expression. We highlight a new mechanism for MMP-1 regulation in human chondrocytes that could provide potential new therapeutic targets.

Synergistic effects of glycoprotein 130 binding cytokines in combination with interleukin-1 on cartilage collagen breakdown.

OBJECTIVE: To determine whether other glycoprotein 130 (gp130) binding cytokines can mimic the effects of oncostatin M (OSM) in acting synergistically with interleukin-1alpha (IL-1alpha) to induce cartilage collagen breakdown and collagenase expression, and to determine which receptors mediate these effects. METHODS: The release of collagen and proteoglycan was assessed in bovine and human cartilage explant cultures. Messenger RNA (mRNA) and protein production from immortalized human chondrocytes (T/C28a4) was analyzed by Northern blotting and specific enzyme-linked immunosorbent assays. Collagenase activity was measured by bioassay. Cell surface receptors were detected by flow cytometry. RESULTS: OSM in combination with IL-1alpha caused a rapid synergistic induction of matrix metalloproteinase 1 mRNA, which was sustained over a 72-hour period. Flow cytometric analyses detected both the OSM-specific receptor and the gp130 receptor at the chondrocyte cell surface, but failed to detect the leukemia inhibitory factor receptor (LIFR). Cartilage degradation assays revealed that, of the gp130 binding cytokines, only OSM and IL-6, in the presence of its soluble receptor (sIL-6R), were able to act synergistically with IL-1alpha to promote collagen release. CONCLUSION: This study demonstrates that IL-6 can mimic OSM in synergizing with IL-1alpha to induce chondrocyte-mediated cartilage collagen breakdown and collagenase production. In order to have this effect, IL-6 requires the presence of its soluble receptor. The apparent absence of LIFR explains why other gp130 binding cytokines do not act in synergy with IL-1alpha. Since OSM, IL-6, and sIL-6R levels have all been shown to be elevated in the rheumatoid joint, our findings suggest that these cytokines may be key mediators of cartilage collagen catabolism in the inflammatory arthritides.

Retinoic acid combines with interleukin-1 to promote the degradation of collagen from bovine nasal cartilage: matrix metalloproteinases-1 and -13 are involved in cartilage collagen breakdown.

Retinoic acid (RetA) and interleukin-1alpha (IL-1) together can induce a reproducible release of proteoglycan fragments from bovine nasal cartilage in culture. However, release of collagen fragments with either agent alone is often variable. In this study over 70% of the total collagen was released from bovine nasal cartilage in culture by day 14 when RetA and IL-1 were combined. This release was accompanied by the appearance of collagenolytic activity in the culture medium that cleaved collagen specifically at the (1/4)/(3/4) position. Tissue inhibitor of metalloproteinases (TIMP) activity was present at day 7 but low or absent in media from resorbing tissue at day 14. The breakdown of cartilage collagen could be prevented by the addition of BB-94, a specific metalloproteinase inhibitor. These results suggest that RetA promotes the early release of TIMP from the tissue and that IL-1 stimulates pro-collagenase secretion which, when activated, exceeds the local concentration of TIMP. Thus in the later stages of culture collagen destruction occurs. Both MMP-1 and MMP-13 were detected and appear to be involved in IL-1 + RetA induced bovine cartilage destruction. However, for the first time, we also present evidence to suggest that MMP-13 is the predominant collagenase in this system.

The role of oncostatin M in animal and human connective tissue collagen turnover and its localization within the rheumatoid joint.

OBJECTIVE: To study the interaction of interleukin-1alpha (IL-1alpha) and oncostatin M (OSM) in promoting cartilage collagen destruction. METHODS: Bovine, porcine, and human cartilage and human chondrocytes were studied in culture. The levels of collagenase (matrix metalloproteinase 1 [MMP-1]) and tissue inhibitor of metalloproteinases 1 (TIMP-1) were measured by bioassay and enzyme-linked immunosorbent assay (ELISA). The levels of OSM in rheumatoid synovial fluid were measured by ELISA. RESULTS: When combined with OSM, IL-1alpha, IL-1beta, and tumor necrosis factor alpha released proteoglycan and collagen from cartilage. OSM was the only member of the IL-6 family to have this effect. Human tendon also responded to IL-1alpha and OSM. OSM increased the production of MMP-1 and TIMP-1 but when combined with IL-1alpha, synergistically promoted MMP-1 production in human chondrocytes and synovial fibroblasts. High levels of OSM were found in human rheumatoid synovial fluids, and confocal microscopy showed that OSM was produced by macrophages in rheumatoid synovial tissue. CONCLUSION: These results highlight an important new mechanism by which there is irreversible loss of collagen from cartilage.

Effects of insulin and insulin-like growth factors I and II on the growth of equine fetal and neonatal chondrocytes.

The effects of insulin and insulin-like growth factors (IGFs) I and II on fetal and foal chondrocytes were investigated in vitro. Chondrocytes from the lateral trochlear ridge of the distal femur were obtained from 2 fetuses (280 and 320 days gestation) and one 4-day-old foal and cultured. Membrane proteins consistent with type 1 and type 2 IGF receptors were demonstrated by radioligand cross linking and equilibrium binding analysis. It was demonstrated that both IGF-I and IGF-II acted as mitogens for isolated equine chondrocytes when present as the sole mitogenic factor in monolayer culture. It was further shown that whereas insulin was able to promote the survival and expansion of cell populations of chondrocytes in culture there was significantly reduced mitogenic stimulation compared to the IGFs. These results suggest that the role of insulin in growth cartilage may be to promote chondrocyte survival, or to suppress differentiation/apoptosis. This supports the hypothesis that relative hyperinsulinaemia may be a contributory factor to equine dyschondroplasia (osteochondrosis). Understanding of contributory, and possibly triggering factors such as this may allow the development of modified methods of husbandry which minimise the risk of disease in populations with a known predisposition.

Cartilage canals in equine articular/epiphyseal growth cartilage and a possible association with dyschondroplasia.

Cartilage canals have been described in most mammals and contain the vascular elements necessary for the maintenance of epiphyseal growth cartilage. The presence and longevity of cartilage canals in developing articular/epiphyseal cartilage of horses is described for the first time. Growth cartilages from 30 normal horses (from 130 days gestation to age 2 years) and 6 cases of dyschondroplasia (age < 15 months) were examined. Cartilage canals were located in the epiphyses of fetuses as early as 130 days gestation. The secondary centres of ossification had developed by 250 days gestation and, at full term (approximately 340 days), the articular/epiphyseal cartilage was highly vascularised. Cartilage canals were closely surrounded by small round chondrocytes even when present in the zone of hypertrophic chondrocytes. From late gestation until at least age 10 weeks, blood vessels surrounded by cartilage were found extending beyond the zone of hypertrophic chondrocytes into the secondary centre of ossification. Cartilage canals were absent from normal articular/epiphyseal cartilage of animals age > 6 months. Cartilage canals were associated with retained cartilage of dyschondroplastic lesions found in animals age < 15 months. The presence of cartilage canals in association with dyschondroplastic lesions suggests they may have a role in the pathogenesis of this important disease.

Collagenase: a key enzyme in collagen turnover.

The primary agents responsible for cartilage and bone destruction in joint diseases are active proteinases that degrade collagen and proteoglycan. All four main classes of proteolytic enzymes are involved in either the normal turnover of connective tissue or its pathological destruction. These proteinases are made by different cells found within the joints. Both extracellular and intracellular pathways exist and individual enzymes can be inhibited by specific proteinaceous inhibitors that block their activity. Recent research has implicated the matrix metalloproteinases (MMPs) in many of the processes involved in joint diseases. The metalloproteinases are capable of degrading all components of the extracellular matrix. This family of proteinases contains a group of at least three collagenases that are capable of degrading native fibrillar collagen. Collagen degradation within joint disease is recognized as the irreversible step in the destruction of cartilage that leads to a failure in joint function. The collagenases are the enzymes necessary to initiate collagen turnover in normal connective tissue turnover and in disease.