R.K. Davidson

Degradome expression profiling in human articular cartilage

IntroductionThe molecular mechanisms underlying cartilage destruction in osteoarthritis are poorly understood. Proteolysis is a key feature in the turnover and degradation of cartilage extracellular matrix where the focus of research has been on the metzincin family of metalloproteinases. However, there is strong evidence to indicate important roles for other catalytic classes of proteases, with both extracellular and intracellular activities. The aim of this study was to profile the expression of the majority of protease genes in all catalytic classes in normal human cartilage and that from patients with osteoarthritis (OA) using a quantitative method.MethodsHuman cartilage was obtained from femoral heads at joint replacement for either osteoarthritis or following fracture to the neck of femur (NOF). Total RNA was purified, and expression of genes assayed using Taqman® low-density array quantitative RT-PCR.ResultsA total of 538 protease genes were profiled, of which 431 were expressed in cartilage. A total of 179 genes were differentially expressed in OA versus NOF cartilage: eight aspartic proteases, 44 cysteine proteases, 76 metalloproteases, 46 serine proteases and five threonine proteases. Wilcoxon ranking as well as the LogitBoost-NR machine learning approach were used to assign significance to each gene, with the most highly ranked genes broadly similar using each method.ConclusionsThis study is the most complete quantitative analysis of protease gene expression in cartilage to date. The data help give direction to future research on the specific function(s) of individual proteases or protease families in cartilage and may help to refine anti-proteolytic strategies in OA.

A gene expression study of normal and damaged cartilage in anteromedial gonarthrosis, a phenotype of osteoarthritis

Summary Objective To identify osteoarthritis (OA) relevant genes and pathways in damaged and undamaged cartilage isolated from the knees of patients with anteromedial gonarthrosis (AMG) – a specific form of knee OA. Design Cartilage was obtained from nine patients undergoing unicompartmental knee replacement (UKR) for AMG. AMG provides a spatial representation of OA progression; showing a reproducible and histologically validated pattern of cartilage destruction such that damaged and undamaged cartilage from within the same knee can be consistently isolated and examined. Gene expression was analysed by microarray and validated using real-time PCR. Results Damaged and undamaged cartilage showed distinct gene expression profiles. 754 genes showed significant up- or down-regulation (non-False discovery rate (FDR) P < 0.05) with enrichment for genes involved in cell signalling, Extracellular Matrix (ECM) and inflammatory response. A number of genes previously unreported in OA showed strongly altered expression including RARRES3, ADAMTSL2 and DUSP10. Confirmation of genes previously identified as modulated in OA was also obtained e.g., SFRP3, MMP3 and IGF1. Conclusions This is the first study to examine a common and consistent phenotype of OA to allow direct comparison of damaged and undamaged cartilage from within the same joint compartment. We have identified specific gene expression profiles in damaged and undamaged cartilage and have determined relevant genes and pathways in OA progression. Importantly this work also highlights the necessity for phenotypic and microanatomical characterization of cartilage in future studies of OA pathogenesis and therapeutic development.

Matrix Metalloproteinase 13 Expression in Response to Double-Stranded RNA in Human Chondrocytes†

OBJECTIVE To investigate the mechanism of matrix metalloproteinase 13 (MMP-13) expression in chondrocytes via pattern-recognition receptors (PRRs) for double-stranded RNA (dsRNA). METHODS Differential expression of PRRs was determined by real-time reverse transcription-polymerase chain reaction (RT-PCR) of RNA from patients with osteoarthritis (OA) and patients with femoral neck fracture (as normal control). Isolated human articular chondrocytes and the chondrosarcoma cell line SW-1353 were activated with poly(I-C) of different molecular weights as a dsRNA mimic, and changes in gene and protein expression were monitored by real-time RT-PCR and immunoblotting, respectively. RESULTS The dsRNA signaling moieties Toll-like receptor 3 (TLR-3), retinoic acid-inducible gene 1 (RIG-1), and nucleotide-binding oligomerization domain-like receptor X1 were all differentially expressed in OA cartilage compared to normal cartilage, as determined by gene expression screening. Depletion of the dsRNA-sensing receptors TLR-3, RIG-1, or melanoma differentiation-associated gene 5 (MDA-5) suppressed the induction of MMP13 messenger RNA (mRNA) expression by poly(I-C), regardless of its mode of delivery. In addition, depletion of the downstream transcription factor interferon regulatory factor 3 resulted in reduced induction of MMP13 mRNA expression by poly(I-C). CONCLUSION Signaling by dsRNA in chondrocytes requires a range of PRRs, including TLR-3, RIG-1, and MDA-5, for the full-induction of MMP13, thus providing tight regulation of a gene critical for maintenance of cartilage integrity. Our data add to the understanding of MMP13 regulation, which is essential before such mechanisms can be exploited to alleviate the cartilage destruction associated with OA.

OP0264 Glucosepane : a new biomarker of the severity of osteoarthritis

Background Glycation, oxidation and nitration of proteins are reactions involved in accelerated ageing of tissues. The products of these reactions are used as biomarkers of chronic pathologies such as diabetes or chronic inflammatory states. Objectives In this work, we studied by mass spectrometry the levels of amino acids and glycated, oxidised or nitrated proteins in culture media of chondrocytes cultivated in multi-layers and in the blood of guinea pigs or osteoarthritis patients. Methods Sixty male, 3-week-old Dunkin-Hartley guinea pigs were used in this work. At 4-weeks-old and 8 week intervals until week 36, twelve animals were sacrificed and histological severity of knee osteoarthritis evaluated and cartilage rheological properties. Human patients with early and advanced osteoarthritis and healthy subjects were recruited. Human chondrocytes cultured in multilayers were treated for 10 days with interleukin (IL) −1β. Amino acids and glycated, oxidised and nitrated proteins were analysed in the serum of guinea pigs, osteoarthritis patients and in the culture medium conditioned by chondrocytes by stable isotopic dilution analysis liquid chromatography-tandem mass spectrometry using the Acquity UPLC system. Results Severity of osteoarthritis increased progressively in guinea pigs with age. Glycated, oxidised and nitrated amino acids were increased markedly at week 36. Glucosepane and dityrosine increased progressively from weeks 20 and 28, respectively. Glucosepane was positively correlated with the OA histological severity (r=0.58, p<0.0001) and the Young’s modulus (r=0.52–0.56, p<0.0001). Oxidation free adducts were positively correlated with OA severity (p<0.0009–0.0029) and hydroxyproline with cartilage thickness (p<0.0003–0.003). In the clinical study, plasma glucosepane was increased 38% in patients with early osteoarthritis (p<0.05) and 6-fold in patients with advanced osteoarthritis (p<0.001) compared to healthy subjects. IL-1β increased the release of glycated, oxidised and nitrated products from chondrocytes in vitro. Conclusions The glycation, oxidation and nitration of proteins are reactions related to the severity of osteoarthritis. The products of these reactions are measurable in blood by mass spectrometry and could be biomarkers of osteoarthritis. More specifically, glucosepane is an advanced glycation product very strongly increased in the severe form of the disease. In conclusion, serum glucosepane is a potential biomarker for diagnosis and progression of osteoarthritis. Disclosure of Interest None declared

DICKKOPF-3 CAN REGULATE CARTILAGE DEGRADATION AND CHONDROCYTE CELL SIGNALLING

Results: The cells without siRNA or CoCl2 treatment were designated as the untreated control cells. The untreated control cells under hypoxia revealed a significant increase in the HIF-1a mRNA levels compared with cells cultured under normoxia. HIF-1a mRNA expression was significantly increased in chondrocytes cultured under simulated hypoxia in comparison with the untreated cells under normoxia. Under condition of hypoxia, si-HIF-1a reduced HIF-1a mRNA levels to about 15.2% at 24hours after transfection, compared to non-targeting scramble siRNA (si-neg) treated cells. The mRNA levels of HSP70, PG, and ColII under hypoxia and simulated hypoxia were significantly increased in comparison with chondrocytes cultured under normoxia. Under condition of hypoxia, the mRNA levels of HSP70, PG, and ColII, were significantly suppressed by si-HIF-1a transfection. Conclusions: Up-regulation of HSP70 under hypoxia is part of the low oxygen stress response seen in Drosophila, and mammalian tissues. Upregulation of heat shock factor (HSF), by which HSP70 are known to be regulated, under hypoxia requires the activity of HIF-1a, the effector of the low oxygen response. In this study, HIF-1a and HSP70 mRNA expressions in cultured chondrocytes were significantly increased under condition of hypoxia and simulated hypoxia. These expressions were not increased in chondrocytes by transfection with si-HIF-1a. These results suggest that HSP70 might be transcriptionally regulated by HSF through HIF-1a in cultured chondrocytes under hypoxia. We previously reported that the expression of HSP70 had an important role for PG and ColII synthesis with heat stimulation in rabbit articular cartilage. In this study, the expression of HSP70, PG, and ColII in cultured chondrocytes were significantly increased under condition of hypoxia, and were increased with CoCl2 under normoxia. But these expressions were not increased under hypoxia in which HIF-1awas inactivated by si-HIF-1a transfection. These findings suggest that PG and ColII gene might be regulated via the HIF-1a and HSP70 pathway under hypoxia.

200 SULFORAPHANE REPRESSES MATRIX-DEGRADING PROTEASES AND PROTECTS CARTILAGE FROM DESTRUCTION IN VITRO

Purpose: Increased expression of inducible NO synthase (iNOS) and cyclooxygenase (COX)-2 plays a key role in the pathogenesis of osteoarthritis (OA). Methylation of lysine 4 on histone H3 (H3K4) was shown to be of fundamental importance in the regulation of gene expression. In the present study, we investigated the role of H3K4 methylation in interleukin-1β (IL-1)-induced COX-2 and iNOS expression in human OA chondrocytes Methods: Chondrocytes were stimulated with IL-1 for various time periods and the expression of iNOS and COX-2 mRNAs and proteins were evaluated using real-time reverse transcriptase-polymerase chain reaction (RT-PCR) and Western blotting, respectively. H3K4 methylation at the iNOS and COX-2 promoters was evaluated using chromatin immunoprecipitation (ChIP) assays. The role of histone methylation was further evaluated using the methyltransferase inhibitor, 5’-deoxy-5’(methylthio) adenosine (MTA). Results: IL-1 induced iNOS and COX-2 mRNA and protein in a doseand time-dependent manner. The induction of iNOS and COX-2 expression by IL-1 was associated with H3K4 diand trimethylation at the iNOS and COX-2 promoters, whereas the levels of H3K4 monomethylation remained unchanged. Treatment with MTA inhibited IL-1-induced H3K4 methylation as well as IL-1-induced iNOS and COX-2 expression. Conclusions: These results indicate that H3K4 methylation contributes to IL-1-induced iNOS and COX-2 expression and suggest that this pathway could be a potential target for pharmacological intervention in the treatment of OA.

048 EVIDENCE FOR A ROLE OF DICKOPPF-3 IN THE PATHOGENESIS OF OSTEOARTHRITIS

Purpose To identify genes showing altered expression in osteoarthritic (OA) cartilage and synovium. Dkk3, a member of the Dickoppf family of Wnt signalling inhibitors was overexpressed and this work highlights the potential function of Dkk3 in OA. Methods Real-time PCR was used to compare the expression of 270 cytokines, chemokines and their receptors in cartilage and synovium from OA and non-OA patients. Expression of Dkk3 was also measured in ATDC5 cells and in bovine nasal cartilage (BNC) explants treated with inflammatory cytokines. The effect of Dkk3 on hydroxyproline and GAG release was measured in BNC explant cultures. To assess the distribution of Dkk3 in OA cartilage immunohistochemistry was carried out on anteromedial gonarthrosis specimens. The level of Dkk3 in synovial fluid tricompartmental and unicompartmental cartilage lesions was measured using ELISA. Results Dkk3 expression was increased 10- in OA cartilage (p=0.00011) and 3.5-fold increase in OA synovium (p=0.007) when compared to respective control tissues. Dkk3 expression was shown to decrease during chondrogenic differentiation of ATDC5 cells and to be increased by interleukin 1 and oncostatin-M in BNC explants. Dkk3 inhibited the release of hydroxyproline and proteoglycan from BNC explants co-treated with interleukin-1 and oncostatin-M. Immunohistochemistry of anteromedial gonarthrosis specimens demonstrated increased Dkk3 in superficial zone chondrocytes in damaged compared to undamaged cartilage from within the same knee. Increased Dkk3 protein was found in the synovial fluid of individuals with tricompartmental OA (n=4) versus unicompartmental cartilage lesions (n=10) (182ng/ml v 116 ng/ml, p Conclusions Dkk3 is a molecule with poorly ascribed function, especially within the musculoskeletal system. In contrast to other members of the Dkk family, Dkk3 does not act consistently as a Wnt inhibitor. Literature on a number of tumour-derived cells have shown that Dkk3 can regulate Wnt, TGFβ, BMP, FGF and Activin signaling and cell proliferation and apoptosis. These cellular processes are highly relevant to OA. In this preliminary study we have shown that Dkk3 is overexpressed in OA cartilage and synovial tissues The decreased expression of Dkk3 during chondrogenesis, and its increase in inflammatory cytokine stimulated BNC explants is suggestive of a role of Dkk3 not only in articular cartilage maintenance but also in development. The ability of Dkk3 to regulate collagen and proteoglycan breakdown (hallmarks of OA) is further evidence for a role in OA pathogenesis. Dkk3 is a compelling molecule that shows potential to further our understanding of OA and to be used as a biomarker of disease or as a target in OA therapeutics.

A22 WHOLE DEGRADOME EXPRESSION PROFILING IN NORMAL AND OSTEOARTHRITIC CARTILAGE

Purpose: Cartilage destruction in osteoarthritis (OA) is thought to be mediated by extracellular proteases. The main focus of research to date has been on the role of two enzyme families; the matrix metalloproteases (MMPs) with respect to cartilage collagen breakdown, and enzymes from the ‘a disintegrin and metalloprotease domain with thrombospondin motifs’ (ADAMTS) family with respect to cartilage aggrecan loss. It is now known that there are around 561 proteases in the human genome (the ‘degradome’): 21 aspartyl, 148 cysteine, 186 metallo-, 178 serine and 28 threonine proteases. The expression of the vast majority of these genes in cartilage is unknown. We have therefore profiled the complete degradome in cartilage from patients with either OA or fracture to the neck of femur (NOF), giving a more complete picture of proteolysis in this tissue and disease. Methods: Cartilage was collected at total hip replacement surgery and snap frozen in liquid nitrogen. Cartilage was ground under liquid nitrogen in a freezer mill and RNA purified by a combination of TRIzol (Invitrogen) and RNeasy (Qiagen). RNA was reverse transcribed and gene expression assessed by quantitative real-time PCR using Taqman low density arrays on the ABI Prism 7900. Results: Expression of 560 proteases and inhibitor genes was detectable in cartilage using this methodology. Preliminary analysis shows that approximately 197 proteases are differentially expressed in OA cartilage compared to NOF at a statistical significance of p< 0.05 (79 metallo-, 50 serine-, 41 cysteine-, 6 aspartyl-, 2 threonine proteases and 19 protease inhibitors). At a significance of p< 0.0001, there were 3 aspartyl, 14 cysteine, 15 metallo-, 12 serine proteases and 5 inhibitors which are differentially regulated in OA. Conclusions: This is the most comprehensive analysis of protease gene expression in cartilage to date. It has identified several protease genes not previously reported to be expressed or regulated in cartilage and provides the foundation on which to build functional studies.