Identification of the pathogenic pathways in osteoarthritic hip cartilage: commonality and discord between hip and knee OA.

OBJECTIVE: To define for the first time the transcriptomes of normal and end-stage osteoarthritis (OA) hip cartilage. MATERIALS AND METHODS: RNA was isolated from cartilage within 2h of joint replacement surgery. Gene expression was analyzed using Agilent GeneSpring GX 11 following hybridization to Illumina Human HT-12 V3 microarrays. Real-time reverse-transcription polymerase chain reaction (RT-PCR) was used to validate the expression of six genes identified by microarray as differentially expressed. Gene Set Enrichment Analysis (GSEA) and Ingenuity Pathway Analysis (IPA) were used to investigate enriched functions or canonical pathways amongst differentially expressed genes respectively. RESULTS: In total we identified 998 differentially expressed genes (fold change ≥ ±1.5, P-value ≤ 0.01) between neck of femur fracture (NOF) (n = 10) and OA hip (n = 9) patient cartilage. These differentially expressed genes were enriched within 71 canonical pathways. A comparison between a comparable knee dataset(20) only identified 229 genes similarly differentially expressed although remarkably 34 canonical pathways overlapped between experiments. CONCLUSIONS: This study is the first to report a comprehensive gene expression analysis of human hip OA cartilage compared to control (NOF) cartilage at the whole-genome level. Our differential gene expression dataset shows excellent correlation with similar defined studies using comparable tissue but reveals discord between hip and knee OA at the individual gene status but with commonality with regards the molecular pathways involved.

Differential Toll-like receptor-dependent collagenase expression in chondrocytes.

OBJECTIVES: To characterise the catabolic response of osteoarthritic chondrocytes to Toll-like receptor (TLR) ligands. METHODS: Induction of the collagenases, matrix metalloproteinase (MMP)1 and MMP13, by TLR ligands was assessed in chondrocytes by real-time reverse transcriptase (RT)-PCR. TLR signalling pathway activation and their involvement in collagenase induction were confirmed by immunoblotting and use of pathway inhibitors and siRNA. TLR expression was compared in the femoral head cartilage of normal controls and patients with osteoarthritis (OA) by real-time RT-PCR. RESULTS: Ligands for TLR6/2 and TLR3 showed the greatest upregulation of MMP1 and MMP13 respectively, although all TLR ligands upregulated these MMPs. MMP1 and MMP13 induction by TLR3 and TLR1/2 or TLR6/2 ligands were dependent on Trif and MyD88, respectively. These inductions were dependent upon the nuclear factor (NF)kappaB pathway, but were differentially inhibited by various mitogen-activated protein kinase inhibitors, with MMP13 induction most reliant on the extracellular signal-regulated kinase pathway. In addition, ligands for TLR1/2 and TLR6/2, but not TLR3, induced significant collagenolysis in a cartilage resorption assay. Finally, TLR2 was significantly downregulated and TLR3 upregulated in OA, compared to normal, cartilage. CONCLUSIONS: Activation of chondrocyte TLRs leads to differential collagenase gene activation. Treatment of chondrocytes with TLR1/2 or TLR6/2 ligands resulted in collagen resorption. The modulated expression of chondrocyte TLR2 and TLR3 in OA cartilage, compared to normal, may reflect a response to repair cartilage or prevent further extracellular matrix destruction. These data suggest modulation of TLR-mediated signalling as a potential therapeutic strategy for the treatment of OA.

The inhibition of metalloproteinases as a therapeutic target in rheumatoid arthritis and osteoarthritis.

The collagenases of the matrix metalloproteinase family are key enzymes in mediating irreversible cartilage collagen loss in arthritis. Inhibition of these enzymes is, therefore, an important therapeutic target. New approaches to collagenase inhibition include active site inhibitors designed for specific enzymes, inhibition of cell signalling molecules and transcription factors involved in collagenase gene expression, prevention of zymogen activation and induction of natural inhibitor production.

Inhibition of cysteine proteinases by a protein inhibitor from potato.

The inhibitory specificity of a protein from potato tubers that inhibits cysteine proteinases (potato cysteine proteinase inhibitor, PCPI) has been compared with that of chicken egg-white cystatin. Most proteinases that are inhibited by cystatin were also inhibited by PCPI, but the potato inhibitor inhibited stem bromelain and fruit bromelain, which are not inhibited by cystatin, and for which no protein inhibitor of comparable potency has previously been described. In contrast, papaya proteinase IV was unaffected by PCPI as it is by the cystatins, and the exopeptidase, dipeptidyl peptidase I, is inhibited by cystatins, but was unaffected by PCPI. The differences in inhibitory specificity between these proteins may well reflect differences between superfamilies of cysteine proteinase inhibitors.

Stem bromelain: amino acid sequence and implications for weak binding of cystatin.

The amino acid sequence of stem bromelain, the major cysteine proteinase from pineapple stem is described. It shows that the enzyme is a member of the papain superfamily of cysteine proteinases, but is not very closely related to any other known member of this group. The sequence shows mutation or deletion of several residues that have been conserved in cysteine proteinases examined previously, including Asn-175 (papain). We suggest that some of these changes have the effect of altering the active-site geometry of stem bromelain, and that this accounts for the resistance of the enzyme to inhibition by cystatins and E-64[L-3-carboxy-2,3-trans-epoxypropionylleucylamido(4-guanidino)b utane].

Debridement of experimental full-thickness skin burns of rats with enzyme fractions derived from pineapple stem.

A limited in vivo study using 12 rats with full-thickness skin burns injuries was carried out. The animals were treated 24 h postburn with two newly discovered enzyme fractions derived from the stem of the pineapple (Ananas comosus). The results indicated that even debridement of the injury could be effected rapidly (within 4 h). Although the details of enzyme formulation and clinical application have yet to be established, these findings clearly suggest that two enzyme fractions from pineapple stem have potential as non-surgical debriding agents.

A computer simulation approach to assessing therapeutic intervention points for the prevention of cytokine-induced cartilage breakdown.

OBJECTIVE: To use a novel computational approach to examine the molecular pathways involved in cartilage breakdown and to use computer simulation to test possible interventions for reducing collagen release. METHODS: We constructed a computational model of the relevant molecular pathways using the Systems Biology Markup Language, a computer-readable format of a biochemical network. The model was constructed using our experimental data showing that interleukin-1 (IL-1) and oncostatin M (OSM) act synergistically to up-regulate collagenase protein levels and activity and initiate cartilage collagen breakdown. Simulations were performed using the COPASI software package. RESULTS: The model predicted that simulated inhibition of JNK or p38 MAPK, and overexpression of tissue inhibitor of metalloproteinases 3 (TIMP-3) led to a reduction in collagen release. Overexpression of TIMP-1 was much less effective than that of TIMP-3 and led to a delay, rather than a reduction, in collagen release. Simulated interventions of receptor antagonists and inhibition of JAK-1, the first kinase in the OSM pathway, were ineffective. So, importantly, the model predicts that it is more effective to intervene at targets that are downstream, such as the JNK pathway, rather than those that are close to the cytokine signal. In vitro experiments confirmed the effectiveness of JNK inhibition. CONCLUSION: Our study shows the value of computer modeling as a tool for examining possible interventions by which to reduce cartilage collagen breakdown. The model predicts that interventions that either prevent transcription or inhibit the activity of collagenases are promising strategies and should be investigated further in an experimental setting.

Assessment of the clinical significance of gelatinase activity in patients with juvenile idiopathic arthritis using quantitative protein substrate zymography.

OBJECTIVE: To measure gelatinase activities in paired synovial fluid (SF) and serum of patients with juvenile idiopathic arthritis (JIA), and to assess how these activities relate to clinical and laboratory measures of disease activity. METHODS: A quantitative protein substrate zymography method was adapted and validated for use with serum and SF. Bands of activity were measured by densitometry and correlated with standard laboratory indicators of inflammation: erythrocyte sedimentation rate and platelet count. RESULTS: Gelatinase activity was found consistently in patients with JIA, with reproducible, quantified bands of activity corresponding to pro-matrix metalloproteinase-9 (pro-MMP-9), including the neutrophil associated lipocalin complex, and pro- and active forms of MMP-2. Both active MMP-2 and pro-MMP-9 were higher in JIA serum than in controls, though no differences were seen between patients grouped according to age, disease duration, or JIA subtype. However, SF MMP-9 correlated significantly with the laboratory indicators of inflammation, as did the relative level of active MMP-2. CONCLUSIONS: Both MMP-2 and MMP-9 gelatinolytic activities are raised during active JIA and associated with inflammatory activity regardless of age and disease duration, supporting a role for MMPs in the breakdown of joint components from early in disease. These MMPs may be specific markers of active joint destruction linked to inflammatory JIA, MMP-9 as a product of infiltrating cells, and the activation of MMP-2 produced within the joint.

Development of a novel 2D proteomics approach for the identification of proteins secreted by primary chondrocytes after stimulation by IL-1 and oncostatin M.

OBJECTIVES: To develop a proteomics approach to study changes in the secreted protein levels of primary human chondrocytes after stimulation by the pro-inflammatory cytokines interleukin-1 and oncostatin M. METHODS: Using both the primary human articular and bovine nasal chondrocyte-conditioned mediums, methods were investigated to enable the separation of proteins by two-dimensional (2D) gel electrophoresis. Differentially regulated proteins were identified using tandem electrospray mass spectrometery. RESULTS: We discovered that proteoglycans and glycosylaminoglycans (GAGs) secreted by chondrocytes significantly interfered with 2D gel focusing. Several different methods for GAG removal were attempted including enzymic digestion, cetyl pyridinium chloride precipitation and anion exchange in high salt. The anion exchange proved to be the most effective. Even from these initial gels, we were able to identify eight proteins produced by human chondrocytes: matrix metalloproteinase (MMP)-1, MMP-3, YKL40, cyclophilin A, beta2-microglobulin, transthyretin, S100A11, peroxidine 1 and cofilin. MMP-1, MMP-3, YKL40 and cyclophilin A were all identified as processed, smaller peptide fragments. CONCLUSIONS: We were able to develop a novel sample preparation protocol to allow the reproducible sample preparation of secreted proteins from human chondrocytes. From the initial data, we were able to show that at least some of the proteins produced were cleaved to smaller fragments as a result of proteolysis. Therefore, this technique provides valuable information about protein processing which gene-based arrays do not.

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.

Human nasal cartilage responds to oncostatin M in combination with interleukin 1 or tumour necrosis factor alpha by the release of collagen fragments via collagenases.

BACKGROUND: The synergistic degradation of cartilage by oncostatin M (OSM) in combination with either interleukin 1 (IL1) or tumour necrosis factor alpha (TNFalpha) has been previously demonstrated using bovine nasal cartilage (BNC). OBJECTIVES: (a) To investigate if human nasal cartilage (HNC) responds in the same way as BNC to these cytokine combinations, particularly in collagen degradation. (b) To compare the response of human nasal and articular cartilages. METHODS: Collagen release was assessed by measuring the hydroxyproline content of culture supernatants and proteoglycan release by the dimethylmethylene blue assay. Matrix metalloproteinase (MMP)-1, MMP-13, and tissue inhibitor of metalloproteinase 1 release were measured by specific enzyme linked immunosorbent assays (ELISAs), and collagenolytic activity was measured by a bioassay using radiolabelled collagen. RESULTS: OSM in combination with either IL1 or TNFalpha acted synergistically to induce collagenolysis from HNC, with a maximum of 79% collagen release. This degradation strongly correlated with MMP-1 and MMP-13 levels and collagenolytic activity. CONCLUSION: Collagen release from human cartilage is marked and implicates both MMP-1 and MMP-13 in the synergistic degradation of human cartilage by OSM in combination with either IL1 or TNFalpha. HNC responds in the same way as BNC, thus validating the bovine cartilage degradation assay as a model relevant to human disease.

Interleukin-6 signalling in juvenile idiopathic arthritis is limited by proteolytically cleaved soluble interleukin-6 receptor.

OBJECTIVES: Interleukin-6 (IL-6) exerts multiple effects on chondrocytes and fibroblasts within the joint and is associated with disease activity in juvenile idiopathic arthritis (JIA). Although these cells express the ubiquitous signalling receptor for all IL-6-related cytokines, gp130, they do not express a cognate IL-6 receptor. Consequently, IL-6 responses within these cells occur via IL-6 trans-signalling relying on the presence of a soluble receptor (sIL-6R). Levels of sIL-6R in vivo are governed by either proteolytic cleavage (PC) of cognate receptor or by differential sIL-6R mRNA splicing (DS). The aim of this study was to evaluate the contribution of both isoforms to clinical parameters associated with IL-6 signalling in JIA. METHODS: IL-6, sIL-6R and DS-sIL-6R were measured by ELISA in serum and synovial fluid (SF) samples from 86 JIA patients. These data were related to indicators of inflammation-erythrocyte sedimentation rate (ESR) and C-reactive protein (CRP) and compared between patients stratified by subtype, age and disease duration. RESULTS: SF IL-6 significantly correlated with general indicators of activity (ESR and CRP) and SF PC-sIL-6R to a lesser degree with CRP. When the IL-6:sIL-6R ratio was calculated as an indicator of the potential for IL-6 signalling within the joint, 33% of SF samples showed a ratio >1 indicating saturation of sIL-6R by IL-6. Mean DS-sIL-6R levels were 0.71 ng/ml, whereas PC-sIL-6R levels constituted the majority of sIL-6R at 20.89 ng/ml. CONCLUSIONS: IL-6 trans-signalling within the joints of JIA patients is predominantly governed by the presence of PC-sIL-6R, and the data provided suggest that synovial levels of IL-6 and sIL-6R would be sufficient to drive IL-6 responses in chondrocytes and synovial fibroblasts.

Interleukin-1 in combination with oncostatin M up-regulates multiple genes in chondrocytes: implications for cartilage destruction and repair.

OBJECTIVE: To identify the genes up-regulated by interleukin-1 (IL-1) in combination with oncostatin M (OSM) in chondrocytes that may be involved in mechanisms of cartilage repair and degradation. METHODS: Gene microarray and real-time polymerase chain reaction (PCR) experiments were performed using RNA from SW1353 chondrocytes and primary human articular chondrocytes. Sections prepared from murine joints, injected with adenovirus vectors overexpressing IL-1 and/or OSM, were analyzed by immunohistochemistry for selected proteins. RESULTS: The combination of IL-1 and OSM markedly up-regulated the expression of various genes, including matrix metalloproteinases (MMPs), cytokines, chemokines, extracellular matrix components, and genes involved in signal transduction. Real-time PCR confirmed a synergistic induction of several MMPs, activin A, pentraxin 3 (PTX-3), and IL-8. The in vivo findings further indicated that stimulation with IL-1 plus OSM induced protein expression of activin A, PTX-3, and KC (the murine homolog of IL-8), as compared with the changes induced by individual cytokine treatment and unstimulated controls. CONCLUSION: The results confirm that the potent proinflammatory cytokine combination of IL-1 plus OSM synergistically and coordinately up-regulates many genes and several MMPs. Moreover, chondrocytes exhibit a potential repair response following this procatabolic stimulus such that the repair mechanisms are ultimately overwhelmed by degradative processes in the cartilage. This gene-profiling study provides insight into the complex processes that mediate joint disease in the inflammatory arthritides through the coordinated expression of multiple genes.

Matrix metalloproteinase 10 promotion of collagenolysis via procollagenase activation: implications for cartilage degradation in arthritis.

OBJECTIVE: We have previously reported the up-regulation of matrix metalloproteinase 10 (MMP-10) following treatment with the procatabolic stimulus of interleukin-1 (IL-1) and oncostatin M (OSM) in chondrocytes. Although MMP-10 is closely related to MMP-3, little is known about the role of MMP-10 in cartilage catabolism. The purpose of this study was to determine whether MMP-10 is expressed in connective tissue cells and to assess how it may contribute to cartilage collagenolysis. METHODS: MMP gene expression was assessed by real-time polymerase chain reaction using RNA from human articular chondrocytes and synovial fibroblasts stimulated with IL-1 plus OSM or tumor necrosis factor alpha (TNFalpha) plus OSM. Synovial fluid levels of MMP-10 were determined by specific immunoassay. Recombinant procollagenases were used in activation studies. Immunohistochemistry assessed MMP-10 expression in diseased joint tissues. RESULTS: MMP-10 expression was confirmed in both chondrocytes and synovial fibroblasts following stimulation with either IL-1 plus OSM or TNFalpha plus OSM, and MMP-10 was detected in synovial fluid samples from patients with various arthropathies. Exogenous MMP-10 significantly enhanced collagenolysis from IL-1 plus OSM-stimulated cartilage, and MMP-10 activated proMMP-1, proMMP-8, and proMMP-13. Immunohistochemistry revealed the presence of MMP-10 in the synovium and cartilage of an IL-1 plus OSM-induced model of arthritis as well as in samples of diseased human tissues. CONCLUSION: We confirm that both synovial fibroblasts and articular chondrocytes express MMP-10 following treatment with procatabolic stimuli. Furthermore, the detectable levels of synovial fluid MMP-10 and the histologic detection of this proteinase in diseased joint tissues strongly implicate MMP-10 in the cartilage degradome during arthritis. The ability of MMP-10 to superactivate procollagenases that are relevant to cartilage degradation suggests that this activation represents an important mechanism by which this MMP contributes to tissue destruction in arthritis.

Levels of matrix metalloproteinase (MMP)-1 in paired sera and synovial fluids of juvenile idiopathic arthritis patients: relationship to inflammatory activity, MMP-3 and tissue inhibitor of metalloproteinases-1 in a longitudinal study.

OBJECTIVES: To measure levels of the collagenases matrix metalloproteinase (MMP)-1 and -13 in the synovial fluid (SF) and serum of patients with juvenile idiopathic arthritis (JIA), and to correlate these measurements with inflammatory activity, levels of the collagenase activator MMP-3 and the tissue inhibitor of metalloproteinases-1 (TIMP-1). METHODS: Levels of MMP-1, -3, -13 and TIMP-1 were measured in paired SF and serum from 82 JIA patients using enzyme-linked immunsorbent assay and compared between subtypes and patients of different ages and disease durations. These levels were also correlated to the active joint count (AJC) and standard measures of inflammatory activity and therapeutic response, including erythrocyte sedimentation rate (ESR) and platelet count (PLT). RESULTS: MMP-1 was detected in JIA SF and correlated with PLT. MMP-3 levels were high in SF and detectable in serum where they correlated with PLT, ESR and AJC. MMP-13, however, was not detected in SF or serum. No differences were observed between patients grouped by subtype, age or disease duration. MMP-3 contributed the majority of total MMP in SF samples resulting in excess MMP levels over TIMP-1. CONCLUSIONS: MMP-1 is up-regulated in SF concordant with inflammatory activity in JIA. This was true for patients in all JIA subtypes and age groups, suggesting that the capability for degradation of type II collagen is present in early disease, and throughout the disease course. MMP-3 may be important in the activation of collagenases and the saturation of exogenous inhibitors. Serum MMP-3 may therefore be a useful, measurable and specific marker of active disease in JIA.

Oncostatin M in combination with tumour necrosis factor {alpha} induces a chondrocyte membrane associated aggrecanase that is distinct from ADAMTS aggrecanase-1 or -2.

OBJECTIVE: To determine whether oncostatin M (OSM) + tumour necrosis factor alpha (TNFalpha) induces aggrecanase activity in chondrocyte membranes, to determine the effects of transforming growth factor beta1 (TGFbeta1), interleukin 4 (IL4), and tissue inhibitor of metalloproteinases (TIMPs) on this activity, and to determine whether this activity is due to a known ADAMTS aggrecanase. METHODS: Aggrecanase activity and ability of agents to prevent membrane associated aggrecanase activity were assessed by Western blotting. Expression of known aggrecanases was measured by real time polymerase chain reaction in bovine nasal and human articular chondrocytes. RESULTS: Chondrocyte membrane associated aggrecanase activity and increased mRNA expression of ADAMTS-1, -4, -5, and -9, but not ADAMTS-4 or -15, were enhanced after stimulation by OSM+TNFalpha in bovine chondrocytes. This activity was inhibited by TIMP-3. In human chondrocytes, OSM+TNFalpha also enhanced ADAMTS-1 and -4 expression, but not that of other ADAMTSs. TNFalpha alone induced ADAMTS-9 expression, whereas OSM addition caused suppression. Both TGFbeta1 and IL4 blocked membrane associated aggrecanase activity and decreased OSM+TNFalpha-induced expression of ADAMTS-9 in bovine and human chondrocytes. IL4 down regulated ADAMTS-4 mRNA, whereas TGFbeta1 increased this expression in both bovine and human chondrocytes. CONCLUSIONS: OSM+TNFalpha up regulates membrane associated aggrecanase activity and several ADAMTS aggrecanase mRNAs in chondrocytes. The chondroprotective effects of IL4 and TIMP-3 suggest that they may have therapeutic benefit for aggrecanolysis, whereas the differential inhibitory effects of TGFbeta1 may limit its therapeutic potential. Induced membrane associated aggrecanase activity is distinct from known soluble ADAMTS aggrecanases and merits further investigation.