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Pleiotropic roles of metallothioneins in osteoarthritis(oa) pathogenesis
Abstracts / Osteoarthritis and Cartilage 25 (2017) S76eS444
a culture condition reducing the inflammatory stress and facilitating investigations with human chondrocytes. 229 REGULATION OF aVb3 AND aVb5 INTEGRIN COMPLEXES BY OSTEOGENIC DIFFERENTIATION OF HUMAN OA HIP CHONDROCYTES E. Charlier, S. Neuville, B. Relic, C. Deroyer, O. Malaise, W. Kurth, P. Gillet, N. Withofs, R. Hustinx, D. de Seny, M.G. Malaise. CHU de Li ege, Li ege, Belgium Purpose: Osteoarthritis (OA) is a chronic degenerative joint disease. It mainly affects cartilage, which is progressively eroded. Osteophytes, or bone spurs, are also common features that form early in OA in an attempt to compensate for joint damage. The cartilage enhances an ossification process to the detriment of the fibrous membrane surrounding bones. In a previous work, we have observed that patients subjected to 18F-FPRGD2 positron emission tomography combined with computed tomography (PET/CT) for oncologic purposes also presented 18 F-FPRGD2 uptake in OA osteoarticular regions (notably in hips and in vertebral osteophytes). avb3 and aVb5 integrin complexes are the main targets of 18F-FPRGD2 radiopharmaceutical. We have previously observed that avb3 was increased in vitro during OA chondrocytes dedifferentiation. In this work, we have investigated whether avb5 integrin was also increased upon dedifferentiation and whether dedifferentiated-like chondrocytes could be found in situ in OA cartilage. We also analysed the osteogenic differentiation capacity of dedifferentiated chondrocytes and its impact on integrin expression. Methods: Human chondrocytes were isolated from OA hip cartilage samples and cultivated in a monolayer for 14 days or more (P1 or P2). Phenotyping experiments were performed by FACS using PE coupledanti avb5 integrin complex. Type III collagen staining was visualized by IHC experiment using normal vs OA cartilage slices. Osteogenic differentiation was induced by culturing OA dedifferentiated chondrocytes with DMEM supplemented with 10 mM b-glycerophosphate, 60 mM ascorbic acid and 10-7 M dexamethasone, for 3 weeks. Medium was replaced every 3e4 days. The induction of alkaline phosphatase (ALP) expression was detected after 3 weeks using histochemical staining assays. The expression of separate integrin subunits was evaluated by western blotting (WB) experiment using specific antibodies. Results: Phenotyping experiment showed that avb5 integrin complex was detected on dedifferentiated chondrocytes (day 14), but not on freshly isolated chondrocytes (day 1) surface (n ¼ 4). Immunohistochemistry experiment showed positive staining for type III collagen in the lacunae located in the upper layers of OA cartilage slice. By contrast, very weak staining was detected in normal cartilage slices. To test the osteogenic potential of OA dedifferentiated chondrocytes (P1 or P2), they were cultivated with or without osteogenic differentiation mix for 3 weeks. Osteogenic mix provoked an increase in dedifferentiated chondrocytes ALP activity, suggesting that these cells display an osteogenic differentiation capacity. Osteogenic differentiation was accompanied by the increase of separate integrin subunits expression (aV, b3 and b5) in cell lysates as observed by WB. Conclusions: aVb5 complex is increased during human OA chondrocytes dedifferentiation in vitro. Type III collagen staining was observed in OA cartilage suggesting the presence of dedifferentiatedlike chondrocytes within OA cartilage. OA dedifferentiated chondrocytes displayed osteogenic differentiation potential. Osteogenic differentiation mix increased separate integrins subunits (aV, b3 and b5) in dedifferentiated chondrocytes lysates. 230 UPREGULATION OF ATROGIN-1/FBXO32 IS NOT NECESSARY FOR CARTILAGE DESTRUCTION IN MOUSE MODELS OF OSTEOARTHRITIS. H.-E. Kim y, z, J. Yang y, z, J. Park y, z, J.-S. Chun y, z. y Gwangju Inst. of Sci. & Technology, Gwangju, Republic of Korea; z Osteoarthritis Res. Ctr., Gwangju, Republic of Korea Purpose: In a preliminary study, we found that recently identified catabolic regulators of osteoarthritis (OA), including hypoxia-inducible factor (HIF)-2a and members of the zinc-ZIP8-MTF1 axis, upregulate the E3 ubiquitin ligase, Atrogin-1 (encoded by Fbxo32), in chondrocytes. As the ubiquitination/proteasomal degradation pathways are tightly regulated to modulate the expression of catabolic factors in chondrocytes, we examined the in vivo functions of Atrogin-1 in mouse models of OA.
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Methods: The mRNA and protein levels of Atrogin-1 and other regulators of OA were determined in primary cultured mouse chondrocytes, OA human cartilage, and OA cartilage from wild-type (WT) and Fbxo32knockout (KO) mice subjected to destabilization of the medial meniscus or intra-articular (IA) injection of adenoviruses expressing HIF-2a (AdEpas1), ZIP8 (Ad-Zip8), or Atrogin-1 (Ad-Fbxo32). The effect of Atrogin1 overexpression on the cartilage of WT mice was examined by IA injection of Ad-Fbxo32. Results: Atrogin-1 mRNA levels in chondrocytes were markedly increased by treatment with interleukin-1b, HIF-2a, and members of the zinc-ZIP8-MTF1 axis. Atrogin-1 protein levels were also increased in OA cartilage from humans and various mouse OA models. However, the forced overexpression of Atrogin-1 in chondrocytes did not modulate the expression of cartilage matrix molecules or matrix-degrading enzymes. Moreover, overexpression of Atrogin-1 in the mouse joint tissues failed to cause OA pathogenesis, and Fbxo32 knockout failed to affect post-traumatic OA cartilage destruction in mice. Conclusions: Although Atrogin-1 is upregulated in OA cartilage, overexpression of Atrogin-1 in the joint tissues or knockout of Fbxo32 does not affect OA cartilage destruction in mice. 231 UPREGULATION OF LIPOCALIN-2 (LCN2) IN OSTEOARTHRITIC CARTILAGE IS NOT NECESSARY FOR CARTILAGE DESTRUCTION IN MICE W.-S. Choi y, z, M.-C. Choi y, z, Y.-O. Son y, z, J.-S. Chun y, z. y Gwangju Inst. of Sci. & Technology, Gwangju, Republic of Korea; z Osteoarthritis Res. Ctr., Gwangju, Republic of Korea Purpose: Lipocalin-2 (LCN2) has recently emerged as a novel adipokines involved in variety of physiological and pathophysiological processes. Although recent in vitro cell-based studies suggest that increased LCN2 level in OA may be detrimental, the precise in vivo role of LCN2 in OA progression has been challenging to define. Here, we performed a set of experiments aimed to elucidate in vivo role of LCN2 in the pathogenesis of OA. Methods: The expression of LCN2 were determined at the mRNA and protein levels in primary cultured mouse chondrocytes and in human and mouse OA cartilage. Experimental OA was induced in wild-type or Lcn2 knockout (KO) mice by destabilization of the medial meniscus (DMM) surgery or intra-articular (IA) injection of adenoviruses expressing HIF-2a (Ad-Epas1), ZIP8 (Ad-Zip8). The effect of LCN2 overexpression on the cartilage of wild-type mice was examined by IA injection of Ad-Lcn2. Primary culture mouse chondrocytes were treated with Lcn2-siRNA or were infected with Ad-Lcn2, and gene expression patterns analyzed by using reverse transcription (RT)-PCR. Results: LCN2 mRNA levels in chondrocytes was markedly increased by the pro-inflammatory cytokines, interleukin (IL)-1b and tumor necrosis factor-a (TNF-a), and by previously identified catabolic regulators of OA, such as hypoxia-inducible factor (HIF)-2a and components of the zincZIP8-MTF1 axis. LCN2 protein levels were also markedly increased in human OA cartilage and cartilage from various experimental mouse models of OA. However, overexpression of LCN2 in chondrocytes did not modulate the expression of cartilage matrix molecules or matrixdegrading enzymes. Furthermore, LCN2 overexpression in mouse cartilage via IA injection of Ad-Lcn2 did not cause OA pathogenesis, and Lcn2 KO mice showed no alteration in DMM-induced OA cartilage destruction. Conclusions: Our observation collectively suggest that upregulation of LCN2 in OA cartilage is not sufficient or necessary for OA cartilage destruction in mice. 232 PLEIOTROPIC ROLES OF METALLOTHIONEINS OSTEOARTHRITIS(OA) PATHOGENESIS
IN
Y. Won y, z, Y. Shin y, z, J.-S. Kwak y, z, W.-K. Song y, x, J.-S. Chun y, z. y Gwangju Inst. of Sci. & Technology, Gwangju, Republic of Korea; z Osteoarthritis Res. Ctr., Gwangju, Republic of Korea; x Bio Imaging and Cell Dynamics Res. Ctr., Gwangju, Republic of Korea Purpose: The zinc-ZIP8-MTF1 axis induces metallothionein (MT) expression and is a catabolic regulator of experimental osteoarthritis (OA) in mice. The main goal of the current study was to invesigate the
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Abstracts / Osteoarthritis and Cartilage 25 (2017) S76eS444
functions and underlying molecular mechanisms of MTs in OA pathogenesis. Methods: Experimental OA in mice was induced by destabilisation of the medial meniscus or intra-articular injection of adenovirus carrying a target gene into wild type, Zip8 chondrocytes-specific knockout(KO) mice, Mtf1 chondrocytes-specific KO mice and Mt1/Mt2 double KO mice. Primary cultured mouse chondrocytes were infected with Ad-Mt1 or AdMt2, and gene expression patterns analyzed via microarray and reverse transcription(RT)-PCR. Proteins in human and mouse OA cartilage were identified via immunohistochemistry. Chondrocyte apoptosis was determined using terminal deoxynucleotidyl transferase (TdT)-mediated deoxyuridine triphosphate (dUTP) nick end labelling (TUNEL). Results: MTs were highly expressed in human and mouse OA cartilage. Hypoxia-inducible factor 2a(Hif-2a), nicotinamide phosphoribosyltransferase(Nampt) and several proinflammatory cytokine pathways, as well as the zinc-ZIP8-MTF1 axis were identified as upstream regulators of MT expression. Genetic deletion of Mt1 and Mt2 enhanced cartilage destruction through increasing chondrocyte apoptosis. Unexpectedly, aberrant overexpression of MT2, but not MT1, induced upregulation of matrix-degrading enzymes and downregulation of matrix molecules through nuclear factor-kappa B (NF-kB) and activator protein-1 (AP-1) activation. Conclusions: MTs have a role in antiapoptotic role in post-traumatic OA. However, aberrant and chronic overexpression of MT2 induces an imbalance between chondrocyte anabolic pathways and catabolic pathways, consequently aggravating OA development. This is the first study to investigate the effect of pleiotropic roles of MTs as regulators of chondrocyte apoptosis as well as catabolic and anabolic pathways during OA pathogenesis. 233 FUNCTIONAL EXPRESSION OF PURINERGIC P2X7 RECEPTORS IN RABBIT ARTICULAR CHONDROCYTE H. Tanigawa y, z, T. Maeda y, M. Kubo y, K. Kumagai y, S. Imai y. y Shiga Univ. of Med. Sci., Otsu, Japan; z Kusatsu Gen. Hosp., Kusatsu city, Japan Purpose: ATP is released by articular chondrocytes under physiological and pathological conditions, which activates plasma membrane purinergic (P2Y and P2X) receptors to modulate the cellular function in an autocrine and paracrine manner. It has been reported that various classes of P2X ion channel receptors are expressed, but little is known about the electrophysiolgical effects of extracellular ATP in articular chondrocytes. The present study was designed to investigate electrophysiological and pharmacological properties of P2X7 as well as its physiological and pathophysiological roles in articular chondrocytes. Methods: Rabbit cartilage was collected from the joints of male animals, and freshly isolated chondrocytes were employed for experiments. The chondrocyte was exposed to external solution containing either ATP or Bz-ATP (a potent agonist for P2X7 receptor) and membrane currents were recorded by using whole-cell patch-clamp technique. The current evoked from P2X7 channels was identified as current components sensitive to its specific blockers. Results: Extracellular application of ATP readily and reversibly activated a non-selective cation conductance that exhibited an inwardly rectifying current-voltage relationship. ATP and Bz-ATP activated the membrane current in a concentration-dependent manner with a halfmaximal excitatory concentration (EC50) of 1.04 mM and 1.15 mM, respectively. ATP-induced activation of the membrane current was almost completely abolished by the P2X7-selective antagonists A438079 (300mM) and A740003 (30mM). Conclusions: These findings provide evidence that P2X7 ion channel receptors function in rabbit articular chondrocytes. Since it is known that P2X7 is involved in diverse pathological processes including inflammatory response and cell death, our study suggests P2X7 as a potential therapeutic target for cartilage disease. 234 WNT16 IS UP-REGULATED INFLAMMATION
IN
THE
JOINT
FOLLOWING
B.L. Thomas, G. Nalesso, S.E. Eldridge, C. Pitzalis, F. Dell'accio. Queen Mary Univ. London, London, United Kingdom Purpose: WNT16 is an injury response gene, absent in adult joints and briefly up-regulated in the initial phases following injury. Yet, its
absence results in a worse outcome of disease. WNT16 has a unique ability to buffer the level of canonical WNT pathway activation and prevent excessive activity. In addition, WNT16 up-regulated Lubricin, a joint lubricant essential for healthy joints. In spite of the only transient expression of WNT16 shortly after injury, its functions are essential for the long term outcomes of osteoarthritis (OA). It is yet not understood what triggers WNT16 expression after injury. The expression of WNT16 coincides with a transient inflammatory response in animal models of OA. The objective of this study is to investigate whether such inflammatory response plays a role in the activation/regulation of WNT16. Methods: Human articular chondrocytes (HAC), were treated with inflammatory stimuli, various pathway inhibitors, as well as recombinant WNT proteins and the outcomes assessed by RT-QPCR and western blot. Results: In the destabilisation of the medial meniscus (DMM) model of instability induced OA, WNT16 was up-regulated in both DMM and sham operated knees as compared to non operated controls, although it persisted longer in DMM operated knees compared to sham operated knees. The period of WNT16 expression coincided with the presence of transient synovial inflammation. Suggesting that inflammatory cytokines may play a role in its regulation. In vitro, recombinant IL1-b treatment was sufficient to induce a robust WNT16 up-regulation in human articular chondrocytes. This up-regulation was dependent on activation of both JNK signalling and CamKII signalling as inhibition of these pathways abolished WNT16 expression. Interestingly, WNT16 expression further activated JNK, thereby suggesting a positive feedback loop. Importantly, exogenous WNT16 protected chondrocytes from IL-1 induced proteoglycan loss. Conclusions: WNT16 is up-regulated following IL1 stimulation and inflammation and is involved in modulating the effects of inflammation in the joint. 235 35 SULPHATE INCORPORATION ASSAY AS A NEW TOOL FOR MEASURING EARLY CARTILAGE DEGRADATION FOLLOWING BLOOD EXPOSURE IN VITRO AND IN VIVO IN F8 KO RATS A.E. Pulles y, z, K.R. Christensen x, k, K. Coeleveld y, M. Kjelgaard-Hansen x, R.E. Schutgens z, F.P. Lafeber y, K. Roepstorff ¶, S.C. Mastbergen y. y Dept. of Rheumatology & Clinical Immunology, Univ. Med. Ctr. Utrecht, Utrecht, Netherlands; z VanCreveld Clinic, Univ. Med. Ctr. Utrecht, Utrecht, Netherlands; x Translational Haemophilia Pharmacology, Novo Nordisk A/S, Maaloev, Denmark; k Vet. Disease Biology, Univ. of Copenhagen, Copenhagen, Denmark; ¶ Histology and Bioimaging, Novo Nordisk A/S, Maaloev, Denmark Purpose: Joint damage upon bleeding causes significant morbidity in patients with hemophilia, and adds to joint degeneration after trauma and major joint surgery. Even a single bleed damages cartilage, synovium and bone, but the pathophysiology is not completely understood. A large part of joint degeneration research uses smaller rodent models with histology as the primary outcome measure. However, histological changes take time to develop and are subject to interpretation despite initiatives to harmonize semi-quantitative scores. Determining proteoglycan synthesis rate, by incorporation of radioactive 35Sulphate (35SO24 ) in cartilage, is a sensitive method to detect early cellular changes in cartilage previously applied in human and larger animal models. Isolating cartilage of small animals can be challenging, so a technique to shave off rat’s cartilage was developed and the 35Sulphate incorporation assay applied to study cartilage degradation following blood exposure. Methods: A total of 13 factor VIII knock out (F8 KO) rats were bred and housed at Novo Nordisk A/S, Maaloev, Denmark. After euthanasia, the legs were removed and transported to UMC Utrecht, The Netherlands. In vitro: within 24 hours after euthanasia the cartilage of six healthy F8 KO rats was obtained by shaving off cartilage fragments of the tibia plateau by use of a scalpel. All cartilage explants were then cultured for four days; in addition to culture medium, half of the cartilage samples were cultured with 50% v/v whole blood. After four days proteoglycan synthesis rate was determined by adding 35SO24 to the cultures for four hours. The 35SO24 becomes incorporated in new synthesized proteoglycans. After digesting the cartilage pieces, cetylpyridinium chloride was added to the samples to precipitate the proteoglycans and the amount of radioactivity was measured by liquid scintillation analysis.
a culture condition reducing the inflammatory stress and facilitating investigations with human chondrocytes. 229 REGULATION OF aVb3 AND aVb5 INTEGRIN COMPLEXES BY OSTEOGENIC DIFFERENTIATION OF HUMAN OA HIP CHONDROCYTES E. Charlier, S. Neuville, B. Relic, C. Deroyer, O. Malaise, W. Kurth, P. Gillet, N. Withofs, R. Hustinx, D. de Seny, M.G. Malaise. CHU de Li ege, Li ege, Belgium Purpose: Osteoarthritis (OA) is a chronic degenerative joint disease. It mainly affects cartilage, which is progressively eroded. Osteophytes, or bone spurs, are also common features that form early in OA in an attempt to compensate for joint damage. The cartilage enhances an ossification process to the detriment of the fibrous membrane surrounding bones. In a previous work, we have observed that patients subjected to 18F-FPRGD2 positron emission tomography combined with computed tomography (PET/CT) for oncologic purposes also presented 18 F-FPRGD2 uptake in OA osteoarticular regions (notably in hips and in vertebral osteophytes). avb3 and aVb5 integrin complexes are the main targets of 18F-FPRGD2 radiopharmaceutical. We have previously observed that avb3 was increased in vitro during OA chondrocytes dedifferentiation. In this work, we have investigated whether avb5 integrin was also increased upon dedifferentiation and whether dedifferentiated-like chondrocytes could be found in situ in OA cartilage. We also analysed the osteogenic differentiation capacity of dedifferentiated chondrocytes and its impact on integrin expression. Methods: Human chondrocytes were isolated from OA hip cartilage samples and cultivated in a monolayer for 14 days or more (P1 or P2). Phenotyping experiments were performed by FACS using PE coupledanti avb5 integrin complex. Type III collagen staining was visualized by IHC experiment using normal vs OA cartilage slices. Osteogenic differentiation was induced by culturing OA dedifferentiated chondrocytes with DMEM supplemented with 10 mM b-glycerophosphate, 60 mM ascorbic acid and 10-7 M dexamethasone, for 3 weeks. Medium was replaced every 3e4 days. The induction of alkaline phosphatase (ALP) expression was detected after 3 weeks using histochemical staining assays. The expression of separate integrin subunits was evaluated by western blotting (WB) experiment using specific antibodies. Results: Phenotyping experiment showed that avb5 integrin complex was detected on dedifferentiated chondrocytes (day 14), but not on freshly isolated chondrocytes (day 1) surface (n ¼ 4). Immunohistochemistry experiment showed positive staining for type III collagen in the lacunae located in the upper layers of OA cartilage slice. By contrast, very weak staining was detected in normal cartilage slices. To test the osteogenic potential of OA dedifferentiated chondrocytes (P1 or P2), they were cultivated with or without osteogenic differentiation mix for 3 weeks. Osteogenic mix provoked an increase in dedifferentiated chondrocytes ALP activity, suggesting that these cells display an osteogenic differentiation capacity. Osteogenic differentiation was accompanied by the increase of separate integrin subunits expression (aV, b3 and b5) in cell lysates as observed by WB. Conclusions: aVb5 complex is increased during human OA chondrocytes dedifferentiation in vitro. Type III collagen staining was observed in OA cartilage suggesting the presence of dedifferentiatedlike chondrocytes within OA cartilage. OA dedifferentiated chondrocytes displayed osteogenic differentiation potential. Osteogenic differentiation mix increased separate integrins subunits (aV, b3 and b5) in dedifferentiated chondrocytes lysates. 230 UPREGULATION OF ATROGIN-1/FBXO32 IS NOT NECESSARY FOR CARTILAGE DESTRUCTION IN MOUSE MODELS OF OSTEOARTHRITIS. H.-E. Kim y, z, J. Yang y, z, J. Park y, z, J.-S. Chun y, z. y Gwangju Inst. of Sci. & Technology, Gwangju, Republic of Korea; z Osteoarthritis Res. Ctr., Gwangju, Republic of Korea Purpose: In a preliminary study, we found that recently identified catabolic regulators of osteoarthritis (OA), including hypoxia-inducible factor (HIF)-2a and members of the zinc-ZIP8-MTF1 axis, upregulate the E3 ubiquitin ligase, Atrogin-1 (encoded by Fbxo32), in chondrocytes. As the ubiquitination/proteasomal degradation pathways are tightly regulated to modulate the expression of catabolic factors in chondrocytes, we examined the in vivo functions of Atrogin-1 in mouse models of OA.
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Methods: The mRNA and protein levels of Atrogin-1 and other regulators of OA were determined in primary cultured mouse chondrocytes, OA human cartilage, and OA cartilage from wild-type (WT) and Fbxo32knockout (KO) mice subjected to destabilization of the medial meniscus or intra-articular (IA) injection of adenoviruses expressing HIF-2a (AdEpas1), ZIP8 (Ad-Zip8), or Atrogin-1 (Ad-Fbxo32). The effect of Atrogin1 overexpression on the cartilage of WT mice was examined by IA injection of Ad-Fbxo32. Results: Atrogin-1 mRNA levels in chondrocytes were markedly increased by treatment with interleukin-1b, HIF-2a, and members of the zinc-ZIP8-MTF1 axis. Atrogin-1 protein levels were also increased in OA cartilage from humans and various mouse OA models. However, the forced overexpression of Atrogin-1 in chondrocytes did not modulate the expression of cartilage matrix molecules or matrix-degrading enzymes. Moreover, overexpression of Atrogin-1 in the mouse joint tissues failed to cause OA pathogenesis, and Fbxo32 knockout failed to affect post-traumatic OA cartilage destruction in mice. Conclusions: Although Atrogin-1 is upregulated in OA cartilage, overexpression of Atrogin-1 in the joint tissues or knockout of Fbxo32 does not affect OA cartilage destruction in mice. 231 UPREGULATION OF LIPOCALIN-2 (LCN2) IN OSTEOARTHRITIC CARTILAGE IS NOT NECESSARY FOR CARTILAGE DESTRUCTION IN MICE W.-S. Choi y, z, M.-C. Choi y, z, Y.-O. Son y, z, J.-S. Chun y, z. y Gwangju Inst. of Sci. & Technology, Gwangju, Republic of Korea; z Osteoarthritis Res. Ctr., Gwangju, Republic of Korea Purpose: Lipocalin-2 (LCN2) has recently emerged as a novel adipokines involved in variety of physiological and pathophysiological processes. Although recent in vitro cell-based studies suggest that increased LCN2 level in OA may be detrimental, the precise in vivo role of LCN2 in OA progression has been challenging to define. Here, we performed a set of experiments aimed to elucidate in vivo role of LCN2 in the pathogenesis of OA. Methods: The expression of LCN2 were determined at the mRNA and protein levels in primary cultured mouse chondrocytes and in human and mouse OA cartilage. Experimental OA was induced in wild-type or Lcn2 knockout (KO) mice by destabilization of the medial meniscus (DMM) surgery or intra-articular (IA) injection of adenoviruses expressing HIF-2a (Ad-Epas1), ZIP8 (Ad-Zip8). The effect of LCN2 overexpression on the cartilage of wild-type mice was examined by IA injection of Ad-Lcn2. Primary culture mouse chondrocytes were treated with Lcn2-siRNA or were infected with Ad-Lcn2, and gene expression patterns analyzed by using reverse transcription (RT)-PCR. Results: LCN2 mRNA levels in chondrocytes was markedly increased by the pro-inflammatory cytokines, interleukin (IL)-1b and tumor necrosis factor-a (TNF-a), and by previously identified catabolic regulators of OA, such as hypoxia-inducible factor (HIF)-2a and components of the zincZIP8-MTF1 axis. LCN2 protein levels were also markedly increased in human OA cartilage and cartilage from various experimental mouse models of OA. However, overexpression of LCN2 in chondrocytes did not modulate the expression of cartilage matrix molecules or matrixdegrading enzymes. Furthermore, LCN2 overexpression in mouse cartilage via IA injection of Ad-Lcn2 did not cause OA pathogenesis, and Lcn2 KO mice showed no alteration in DMM-induced OA cartilage destruction. Conclusions: Our observation collectively suggest that upregulation of LCN2 in OA cartilage is not sufficient or necessary for OA cartilage destruction in mice. 232 PLEIOTROPIC ROLES OF METALLOTHIONEINS OSTEOARTHRITIS(OA) PATHOGENESIS
IN
Y. Won y, z, Y. Shin y, z, J.-S. Kwak y, z, W.-K. Song y, x, J.-S. Chun y, z. y Gwangju Inst. of Sci. & Technology, Gwangju, Republic of Korea; z Osteoarthritis Res. Ctr., Gwangju, Republic of Korea; x Bio Imaging and Cell Dynamics Res. Ctr., Gwangju, Republic of Korea Purpose: The zinc-ZIP8-MTF1 axis induces metallothionein (MT) expression and is a catabolic regulator of experimental osteoarthritis (OA) in mice. The main goal of the current study was to invesigate the
S156
Abstracts / Osteoarthritis and Cartilage 25 (2017) S76eS444
functions and underlying molecular mechanisms of MTs in OA pathogenesis. Methods: Experimental OA in mice was induced by destabilisation of the medial meniscus or intra-articular injection of adenovirus carrying a target gene into wild type, Zip8 chondrocytes-specific knockout(KO) mice, Mtf1 chondrocytes-specific KO mice and Mt1/Mt2 double KO mice. Primary cultured mouse chondrocytes were infected with Ad-Mt1 or AdMt2, and gene expression patterns analyzed via microarray and reverse transcription(RT)-PCR. Proteins in human and mouse OA cartilage were identified via immunohistochemistry. Chondrocyte apoptosis was determined using terminal deoxynucleotidyl transferase (TdT)-mediated deoxyuridine triphosphate (dUTP) nick end labelling (TUNEL). Results: MTs were highly expressed in human and mouse OA cartilage. Hypoxia-inducible factor 2a(Hif-2a), nicotinamide phosphoribosyltransferase(Nampt) and several proinflammatory cytokine pathways, as well as the zinc-ZIP8-MTF1 axis were identified as upstream regulators of MT expression. Genetic deletion of Mt1 and Mt2 enhanced cartilage destruction through increasing chondrocyte apoptosis. Unexpectedly, aberrant overexpression of MT2, but not MT1, induced upregulation of matrix-degrading enzymes and downregulation of matrix molecules through nuclear factor-kappa B (NF-kB) and activator protein-1 (AP-1) activation. Conclusions: MTs have a role in antiapoptotic role in post-traumatic OA. However, aberrant and chronic overexpression of MT2 induces an imbalance between chondrocyte anabolic pathways and catabolic pathways, consequently aggravating OA development. This is the first study to investigate the effect of pleiotropic roles of MTs as regulators of chondrocyte apoptosis as well as catabolic and anabolic pathways during OA pathogenesis. 233 FUNCTIONAL EXPRESSION OF PURINERGIC P2X7 RECEPTORS IN RABBIT ARTICULAR CHONDROCYTE H. Tanigawa y, z, T. Maeda y, M. Kubo y, K. Kumagai y, S. Imai y. y Shiga Univ. of Med. Sci., Otsu, Japan; z Kusatsu Gen. Hosp., Kusatsu city, Japan Purpose: ATP is released by articular chondrocytes under physiological and pathological conditions, which activates plasma membrane purinergic (P2Y and P2X) receptors to modulate the cellular function in an autocrine and paracrine manner. It has been reported that various classes of P2X ion channel receptors are expressed, but little is known about the electrophysiolgical effects of extracellular ATP in articular chondrocytes. The present study was designed to investigate electrophysiological and pharmacological properties of P2X7 as well as its physiological and pathophysiological roles in articular chondrocytes. Methods: Rabbit cartilage was collected from the joints of male animals, and freshly isolated chondrocytes were employed for experiments. The chondrocyte was exposed to external solution containing either ATP or Bz-ATP (a potent agonist for P2X7 receptor) and membrane currents were recorded by using whole-cell patch-clamp technique. The current evoked from P2X7 channels was identified as current components sensitive to its specific blockers. Results: Extracellular application of ATP readily and reversibly activated a non-selective cation conductance that exhibited an inwardly rectifying current-voltage relationship. ATP and Bz-ATP activated the membrane current in a concentration-dependent manner with a halfmaximal excitatory concentration (EC50) of 1.04 mM and 1.15 mM, respectively. ATP-induced activation of the membrane current was almost completely abolished by the P2X7-selective antagonists A438079 (300mM) and A740003 (30mM). Conclusions: These findings provide evidence that P2X7 ion channel receptors function in rabbit articular chondrocytes. Since it is known that P2X7 is involved in diverse pathological processes including inflammatory response and cell death, our study suggests P2X7 as a potential therapeutic target for cartilage disease. 234 WNT16 IS UP-REGULATED INFLAMMATION
IN
THE
JOINT
FOLLOWING
B.L. Thomas, G. Nalesso, S.E. Eldridge, C. Pitzalis, F. Dell'accio. Queen Mary Univ. London, London, United Kingdom Purpose: WNT16 is an injury response gene, absent in adult joints and briefly up-regulated in the initial phases following injury. Yet, its
absence results in a worse outcome of disease. WNT16 has a unique ability to buffer the level of canonical WNT pathway activation and prevent excessive activity. In addition, WNT16 up-regulated Lubricin, a joint lubricant essential for healthy joints. In spite of the only transient expression of WNT16 shortly after injury, its functions are essential for the long term outcomes of osteoarthritis (OA). It is yet not understood what triggers WNT16 expression after injury. The expression of WNT16 coincides with a transient inflammatory response in animal models of OA. The objective of this study is to investigate whether such inflammatory response plays a role in the activation/regulation of WNT16. Methods: Human articular chondrocytes (HAC), were treated with inflammatory stimuli, various pathway inhibitors, as well as recombinant WNT proteins and the outcomes assessed by RT-QPCR and western blot. Results: In the destabilisation of the medial meniscus (DMM) model of instability induced OA, WNT16 was up-regulated in both DMM and sham operated knees as compared to non operated controls, although it persisted longer in DMM operated knees compared to sham operated knees. The period of WNT16 expression coincided with the presence of transient synovial inflammation. Suggesting that inflammatory cytokines may play a role in its regulation. In vitro, recombinant IL1-b treatment was sufficient to induce a robust WNT16 up-regulation in human articular chondrocytes. This up-regulation was dependent on activation of both JNK signalling and CamKII signalling as inhibition of these pathways abolished WNT16 expression. Interestingly, WNT16 expression further activated JNK, thereby suggesting a positive feedback loop. Importantly, exogenous WNT16 protected chondrocytes from IL-1 induced proteoglycan loss. Conclusions: WNT16 is up-regulated following IL1 stimulation and inflammation and is involved in modulating the effects of inflammation in the joint. 235 35 SULPHATE INCORPORATION ASSAY AS A NEW TOOL FOR MEASURING EARLY CARTILAGE DEGRADATION FOLLOWING BLOOD EXPOSURE IN VITRO AND IN VIVO IN F8 KO RATS A.E. Pulles y, z, K.R. Christensen x, k, K. Coeleveld y, M. Kjelgaard-Hansen x, R.E. Schutgens z, F.P. Lafeber y, K. Roepstorff ¶, S.C. Mastbergen y. y Dept. of Rheumatology & Clinical Immunology, Univ. Med. Ctr. Utrecht, Utrecht, Netherlands; z VanCreveld Clinic, Univ. Med. Ctr. Utrecht, Utrecht, Netherlands; x Translational Haemophilia Pharmacology, Novo Nordisk A/S, Maaloev, Denmark; k Vet. Disease Biology, Univ. of Copenhagen, Copenhagen, Denmark; ¶ Histology and Bioimaging, Novo Nordisk A/S, Maaloev, Denmark Purpose: Joint damage upon bleeding causes significant morbidity in patients with hemophilia, and adds to joint degeneration after trauma and major joint surgery. Even a single bleed damages cartilage, synovium and bone, but the pathophysiology is not completely understood. A large part of joint degeneration research uses smaller rodent models with histology as the primary outcome measure. However, histological changes take time to develop and are subject to interpretation despite initiatives to harmonize semi-quantitative scores. Determining proteoglycan synthesis rate, by incorporation of radioactive 35Sulphate (35SO24 ) in cartilage, is a sensitive method to detect early cellular changes in cartilage previously applied in human and larger animal models. Isolating cartilage of small animals can be challenging, so a technique to shave off rat’s cartilage was developed and the 35Sulphate incorporation assay applied to study cartilage degradation following blood exposure. Methods: A total of 13 factor VIII knock out (F8 KO) rats were bred and housed at Novo Nordisk A/S, Maaloev, Denmark. After euthanasia, the legs were removed and transported to UMC Utrecht, The Netherlands. In vitro: within 24 hours after euthanasia the cartilage of six healthy F8 KO rats was obtained by shaving off cartilage fragments of the tibia plateau by use of a scalpel. All cartilage explants were then cultured for four days; in addition to culture medium, half of the cartilage samples were cultured with 50% v/v whole blood. After four days proteoglycan synthesis rate was determined by adding 35SO24 to the cultures for four hours. The 35SO24 becomes incorporated in new synthesized proteoglycans. After digesting the cartilage pieces, cetylpyridinium chloride was added to the samples to precipitate the proteoglycans and the amount of radioactivity was measured by liquid scintillation analysis.