Hypoxic resistance to articular chondrocyte apoptosis – a possible mechanism of maintaining homeostasis of normal articular cartilage J.-W Seol1, H.-B Lee1, Y.-J Lee1, Y.-H Lee2, H.-s Kang1, I.-s Kim1, N.-S Kim1 and S.-Y Park1 Center for Healthcare Technology Development, Bio-Safety Research Institute, College of Veterinary Medicine, Chonbuk National University, Jeonju, Jeonbuk, South Korea Institute of Oral Bioscience, School of Dentistry, Chonbuk National University, Jeonju, Jeonbuk, South Korea Keywords chondrocytes; hypoxia; proteasome; reactive oxygen species; tumour necrosis factor-related apoptosis-inducing ligand (TRAIL) Correspondence S.-Y Park, College of Veterinary Medicine, Chonbuk National University, Jeonju, Jeonbuk 561-756, South Korea Fax: +82 63 270 3780 Tel: +82 63 270 3886 E-mail: sypark@chonbuk.ac.kr (Received 21 August 2009, revised 10 October 2009, accepted 20 October 2009) doi:10.1111/j.1742-4658.2009.07451.x Hypoxia and hypoxia-related genes are important factors in articular chondrocytes during cartilage homeostasis and osteoarthritis We have investigated the various apoptotic factors that show significance in synovial fluid obtained from normal and experimental osteoarthritic animal models and have evaluated the effect of hypoxia on articular chondrocyte apoptosis induced by these apoptotic factors Mature beagle dogs underwent surgical transections of ligaments and medial meniscectomies to explore the underlying mechanisms of osteoarthritis Cartilage and synovial fluid obtained from normal animals and those with osteoarthritis were evaluated via proteasome inhibition, tumour necrosis factor-related apoptosis-inducing ligand (TRAIL) protein expression, mitochondrial transmembrane potential and levels of reactive oxygen species Canine chondrocytes were exposed to the proteasome inhibitor N-acetyl-Leu-Leu-Norleu-al and treated with recombinant TRAIL protein under normoxic and hypoxic conditions, measuring chondrocyte cell viability, proteasome activity and levels of apoptotic factors TRAIL protein expression and ubiquitinated proteins were increased significantly, but the proteasome activity in the synovial fluid of osteoarthritic joints relative to that in normal joints was not Primary cultured articular chondrocytes cotreated with the proteasome inhibitor and TRAIL progressed to severe apoptosis under normoxic conditions, but the sensitization caused by the combined treatment was suppressed by exposure to hypoxia Caspase-8 activation, c-Jun N-terminal kinase phosphorylation, the mitochondrial transmembrane potential and the generation of reactive oxygen species involved in cell death regulation were significantly inhibited under hypoxic conditions These findings suggest that proteasome inhibition and TRAIL may be possible mechanisms in cartilage degradation and joint-related diseases Furthermore, the maintenance of hypoxic conditions or therapy with hypoxia-related genes in the joint may be successful for the treatment of joint-related diseases, including osteoarthritis Abbreviations ALLN, N-acetyl-Leu-Leu-Norleu-al; DCFH2-DA, 2¢,7¢-dichlorodihydrofluorescein diacetate; DR-5, death receptor-5; JC-1, 5,5¢,6,6¢-tetrachloro1,1¢3,3¢-tetraethylbenzimidazol-carbocyanine iodide; JNK, c-Jun N-terminal kinase; JNK-SAPK, c-Jun N-terminal kinase-stress-activated protein kinase; MTP, mitochondrial transmembrane potential; OA, osteoarthritis; ROS, reactive oxygen species; Suc-LLVY-AMC, Suc-Leu-Leu-ValTyr-7-amino-4-methylcoumarin; TRAIL, tumour necrosis factor-related apoptosis-inducing ligand FEBS Journal 276 (2009) 7375–7385 ª 2009 The Authors Journal compilation ª 2009 FEBS 7375 Hypoxic condition inhibits articular chondrocyte death J.-W Seol et al Introduction A hallmark of osteoarthritis (OA) is a decrease in the number of chondrocytes, as they are the only resident cells in articular cartilage Chondrocytes regulate the enzymatic breakdown of the extracellular matrix, thereby maintaining the equilibrium between synthetic and degradative processes in the cartilage [1] Therefore, the metabolic and structural changes of chondrocytes in the articular cartilage play a significant role in the initiation and progression of the disease Several studies have examined cell death in human articular cartilage affected by OA or in experimental models of OA [2,3] Tumour necrosis factor-related apoptosis-inducing ligands (TRAILs) are type II transmembrane molecules that trigger the apoptotic signal cascade by binding to cognate receptors expressed on the cell surface [4,5] TRAIL is highly expressed on the surfaces of natural killer (NK) cells, as well as on CD4+ and CD8+ T cells It promotes apoptosis, which may aid in the resolution of infection and the attenuation of the development of streptoxotocin-induced diabetes and collagen-induced arthritis [6–9] Some studies have reported a role for TRAIL protein in articular joint disease [10,11] TRAIL alone can induce apoptosis in primary cultured chondrocytes from different animal species, such as humans and rats [11,12], but the exact role of TRAIL in chondrocytes has not been clearly defined to date Ubiquitin-proteasome-mediated protein degradation pathways have been shown to play an important role in regulating both cell proliferation and cell death [13] Most recent studies have suggested that ubiquitin-proteasome-dependent proteolysis is also involved in apoptosis, although its exact role remains controversial [14] Proteasome inhibitors block the process of programmed cell death in thymocytes and neurons, but induce apoptosis in various human cancer cell lines Proteasome inhibition suppresses growth plate proliferation and induces chondrocyte apoptosis [15] Human chondrocytes are also sensitive to proteasome-induced apoptosis [16] Although the treatment of cells with this compound causes marked increases in a large number of cellular proteins, including cyclin-dependent protein kinase inhibitors, it is not clear how this agent actually induces apoptosis [17,18] More research is required to fully characterize the types of cell death in aging and arthritic cartilage, together with their respective frequencies Articular cartilage is an avascular tissue that functions at an oxygen tension lower than that of most other tissues, and derives both its nutrition and oxygen 7376 supply by diffusion from the synovial fluid and subchondral bone [19,20] It has been estimated that articular chondrocytes in the deepest layers may have access to no more than 1–6% O2 [21,22] Oxygen can be processed to generate reactive oxygen species (ROS), which play an important role in intracellular signalling and thus in cell physiology and cellular destruction ROS are known to induce a wide range of responses, depending on cell type and levels of ROS within the cell [23,24] The aims of this study were to investigate the major signalling pathways and effects of hypoxic conditions in experimental osteoarthritic cartilage degeneration and cell death of primary cultured chondrocytes In particular, we focused on the role of proteasome inhibition and TRAIL in osteoarthritic disease and chondrocyte apoptosis, and the hypoxic inhibition of cartilage and chondrocyte degeneration Results Macroscopic and radiographic examination of the articular cartilage after experimentally induced OA Articular cartilage of the femoral condyles from the experimental joints was examined to assess any macroscopic damage caused by experimentally induced OA Cartilage damage was visualized on the tibial plateau of the experimental joints when compared with normal control joints The medial tibia plateau cartilage in the experimental joints was fibrillated with erosive lesions (Fig 1A), and radiographic findings revealed joint distension There was no evidence of sclerosis, erosions or osteophyte and enthesophyte formation in the experimental joints (Fig 1B) TRAIL and ubiquitinated protein expression were significantly increased, but proteasome activity was not, in the synovial fluid of osteoarthritic joints Proteasome activity was assayed in the synovial fluid from experimental osteoarthritic joints via Suc-LeuLeu-Val-Tyr-7-amino-4-methylcoumarin (Suc-LLVYAMC) hydrolysis, and was significantly lower than the activity in control joints The ubiquitinated protein levels in synovial fluid from osteoarthritic joints were higher than in the control joint group (Fig 2A) We also examined TRAIL protein expression in experimental osteoarthritic synovial fluid The protein FEBS Journal 276 (2009) 7375–7385 ª 2009 The Authors Journal compilation ª 2009 FEBS J.-W Seol et al Hypoxic condition inhibits articular chondrocyte death A A B B Fig Evaluation of articular cartilage after experimentally induced osteoarthritis (A) Photomicrographs of articular cartilage (B) The evaluation of osteoarthritis in the right and left joints of dogs was graded 12 weeks after surgery by the evaluation of radiographs using established parameters OA, osteoarthritis sample expression of TRAIL was increased in osteoarthritic joints compared with the control group (Fig 2B) In this experiment, the elevated TRAIL protein of osteoarthritic synovial fluid may have originated from various inflammatory cells, such as lymphocytes, and other studies support this [6,8] Combined treatment with proteasome inhibitor and TRAIL markedly enhanced apoptosis in cultured canine chondrocytes To investigate the proteasome inhibition effect on TRAIL-induced apoptosis in canine chondrocytes, N-acetyl-Leu-Leu-Norleu-al (ALLN) was used as a proteasome inhibitor Canine chondrocytes were exposed to ALLN (10 lm) for 12 h and then treated with recombinant TRAIL protein for an additional 12 h TRAIL and ALLN alone did not induce apoptosis, but combined treatment markedly induced apoptosis to 60% in canine chondrocytes (Fig 3A) The examination of cell morphology also supported the enhancing effect of combined ALLN and TRAIL treatment in canine chondrocytes (Fig 3B) To determine whether treatment with the proteasome inhibitor ALLN affected proteasome-mediated Fig TRAIL protein and ubiquitinated protein levels were significantly higher and proteasome activity was lower in osteoarthritic joints (A) Proteasome activity was measured using the synthetic fluorogenic substrate Suc-LLVY-AMC Fluorescence was measured at 380 nm excitation and 440 nm emission The fluorescence value for control cells was set at 100%, and the fluorescence values relative to the control are presented The experiments were performed in triplicate at least twice independently (A, B) Proteins were separated on an 8–15% SDS gel, and apoptotic proteins were detected by western blot analysis b-Actin was used to normalize equal protein loading Blot images represent one of three independent experiments *P < 0.05 versus normal sample was calculated using Student’s t-test OA, osteoarthritis sample; Ubi, ubiquitin degradation in canine chondrocytes, proteasome activity was assayed in cell lysates as a measure of the hydrolysis of the fluorogenic substrate Suc-LLVYAMC Proteasome activity was significantly inhibited by treatment with ALLN only and cotreatment with ALLN and TRAIL (Fig 3C) Western blot analysis was also used to investigate whether ALLN induced proteasome inhibition in canine chondrocytes In the absence of ALLN, smears of ubiquitinated proteins were not observed in control and TRAIL-treated canine chondrocytes In the presence of ALLN, marked accumulation of polyubiquitinated proteins was observed in canine chondrocytes (Fig 3D) Proteasome inhibition increased significantly TRAIL-mediated caspase-8 activation and JNK phosphorylation To determine the mechanism by which proteasome inhibition enhanced TRAIL-induced apoptosis in canine chondrocytes, we examined caspase-8 activation and death receptor-5 (DR-5) and TRAIL protein FEBS Journal 276 (2009) 7375–7385 ª 2009 The Authors Journal compilation ª 2009 FEBS 7377 Hypoxic condition inhibits articular chondrocyte death J.-W Seol et al A C B D Fig Proteasome inhibition markedly enhanced TRAIL-induced apoptosis and significantly inhibited proteasome activity in primary cultured canine chondrocytes (A) Cell viability was determined by the crystal violet staining method The viability of control cells was set at 100%, and the viability relative to the control is presented The experiments were performed in triplicate at least twice independently The bars describe the standard deviation (B) Cell morphology was photographed (·200) under the various conditions (C) Proteasome activity was measured using the synthetic fluorogenic substrate Suc-LLVY-AMC Fluorescence was measured at 380 nm excitation and 440 nm emission The fluorescence value for control cells was set at 100%, and the fluorescence values relative to the control are presented The experiments were performed in triplicate at least twice independently The bars describe the standard deviation (D) Whole-cell lysates were prepared and total protein (40 lgỈmL)1) was electrophoretically resolved on SDS gel Ubiquitin protein levels were detected by western blotting analysis b-Actin was used to normalize equal protein loading Blot images represent one of three independent experiments **P < 0.01, *P < 0.05 versus control were calculated using Student’s t-test expression in cells treated with ALLN and ⁄ or TRAIL protein Western blot analysis showed that caspase-8 was slightly activated in control, ALLN-treated and TRAIL-treated chondrocytes However, under proteasome inhibition induced by pretreatment with ALLN, TRAIL treatment unexpectedly increased the activation of caspase-8 In addition, the phosphorylation of JNK protein was markedly increased by combined treatment with ALLN and TRAIL when compared with other groups The expression of DR-5 and TRAIL protein was also investigated, but these were not altered in cells with proteasome inhibition (Fig 4) Proteasome inhibition and the expression of TRAIL protein induced the dissipation of the mitochondrial transmembrane potential (MTP) and ROS generation MTP was investigated in order to address the possible mechanism by which proteasome inhibition enhances TRAIL-induced apoptosis in canine chondrocytes 7378 MTP evaluation is based on the ability of a fluorescent probe to enter the mitochondria selectively and reversibly change its colour from green to red as the mitochondrial potential increases 5,5¢,6,6¢-Tetrachloro1,1¢3,3¢-tetraethylbenzimidazol-carbocyanine iodide (JC-1; Molecular Probes, Eugene, OR, USA) exists as a monomer at low MTP values and shows green fluorescence, whereas it forms an aggregate at high MTP and shows red fluorescence The fluoroscopic results presented in Fig 5A show a red and slightly green fluorescence in cells treated with ALLN and TRAIL alone, but a highly green fluorescence after combined treatment Photomicrographs indicated that ALLN and TRAIL alone induced a small change in MTP, whereas combined treatment with ALLN and TRAIL caused a significant dissipation of MTP relative to negative controls When ROS generation was examined, the results showed that pretreatment of the cells with the proteasome inhibitor increased ROS levels, and that significant ROS generation was induced with TRAIL cotreatment in canine chondrocytes (Fig 5B) FEBS Journal 276 (2009) 7375–7385 ª 2009 The Authors Journal compilation ª 2009 FEBS J.-W Seol et al Hypoxic condition inhibits articular chondrocyte death To investigate the effects of MTP in cartilage by the induction of OA, chondrocytes were isolated from experimentally induced osteoarthritic cartilage The photomicrographs and fluorescence values indicated that these chondrocytes showed a decrease in MTP compared with normal cartilage (Fig 5C) In addition, the chondrocytes isolated from osteoarthritic joints demonstrated significantly greater ROS generation than did the controls (Fig 5D) Hypoxia inhibited the apoptosis of primary cultured canine chondrocytes induced by proteasome inhibition and TRAIL treatment Fig Proteasome inhibition and TRAIL treatment significantly increased caspase-8 activation and JNK phosphorylation Whole-cell lysates were prepared and total protein (40 lgỈmL)1) was electrophoretically resolved on SDS gel Apoptotic proteins were detected by western blotting analysis b-Actin was used to normalize equal protein loading Blot images represent one of three independent experiments In order to examine the functional role of ALLN and TRAIL in apoptotic cell death under hypoxic conditions, canine chondrocytes were exposed to hypoxia and ALLN (10 lm) for 12 h, and were then treated with recombinant TRAIL protein for an additional 12 h under normoxic and hypoxic conditions Hypoxic conditions inhibited significantly the apoptosis of chondrocytes induced by cotreatment of the cells with the proteasome inhibitor and TRAIL (Fig 6A) Chondrocyte survival under hypoxic conditions was enhanced by 25% compared with the survival of cells A C B D Fig The decrease in MTP and ROS generation induced by proteasome inhibition and TRAIL (A, C) MTP was determined using a JC-1 probe The cells were photographed using a fluoroscope The green fluorescence intensity was measured under the conditions described in Materials and methods The experiments were performed in triplicate at least twice independently (B, D) The ROS level was measured using DCFH-DA DCFH fluorescence was determined with a fluorescence plate reader with 490 and 525 nm as excitation and emission wavelengths, respectively The fluorescence value for control cells was set at 100%; fluorescence values relative to the control are presented The experiments were performed in triplicate at least twice independently **P < 0.01, *P < 0.05 versus control were calculated using Student’s t-test MFI, mean fluorescence intensity; OA, osteoarthritis sample FEBS Journal 276 (2009) 7375–7385 ª 2009 The Authors Journal compilation ª 2009 FEBS 7379 Hypoxic condition inhibits articular chondrocyte death J.-W Seol et al A C B D Fig Hypoxia inhibited chondrocyte death and apoptosis-related signals induced by proteasome inhibition and TRAIL (A) Cell viability was determined using the crystal violet staining method The control cell viability was set at 100%; viability relative to the control is presented The experiments were performed in triplicate at least twice independently The cell morphology was photographed (·200) (B) Whole-cell lysates were prepared and total protein (40 lgỈmL)1) was electrophoretically resolved on SDS gel and then tested for apoptotic proteins by western blotting analysis b-Actin was used to normalize equal protein loading; Blot images represent one of three independent experiments (C) MTP was determined using a JC-1 probe The cells were photographed using a fluoroscope The green fluorescence intensity was measured under the conditions described in Materials and methods (D) The ROS level was measured using DCFH-DA The fluorescence value for control cells was set at 100%; fluorescence values relative to the control are presented The experiments were performed in triplicate at least twice independently **P < 0.01, *P < 0.05 versus control were calculated using Student’s n-test Nor, normoxia; Hypo, hypoxia; A, ALLN; T, TRAIL; MFI, mean fluorescence intensity that were cotreated with ALLN and TRAIL under normoxic conditions Moreover, photomicrographs revealed that cells showed a decreased death rate under hypoxic conditions when they were cotreated with ALLN and TRAIL (Fig 6A) Hypoxia inhibited chondrocyte apoptosis through the inhibition of caspase activation, JNK phosphorylation, restoration of MTP loss and ROS generation To examine why hypoxia inhibited the combined effects of ALLN and TRAIL in canine chondrocytes, western blot analysis was performed It was shown that cotreatment of cells with ALLN and TRAIL increased caspase-8 activation and JNK phosphorylation However, both caspase-8 activation and JNK 7380 phosphorylation were inhibited under hypoxic conditions (Fig 6B) MTP and ROS were investigated in order to address the inhibitory mechanism exerted by hypoxic conditions Canine chondrocytes were exposed to ALLN (10 lm) for 12 h and were then treated with recombinant TRAIL protein for an additional 12 h under normoxic and hypoxic conditions The fluoroscopic results presented in Fig 6C show that the cells fluoresce green after cotreatment with ALLN and TRAIL, indicating lower MTP under normoxic conditions However, the green fluorescence indicating lower MTP declined under hypoxic conditions (Fig 6C) Cotreatment of cells with ALLN and TRAIL induced ROS generation significantly under normoxic conditions, but hypoxia prevented ROS generation after cotreatment with ALLN and TRAIL (Fig 6D) FEBS Journal 276 (2009) 7375–7385 ª 2009 The Authors Journal compilation ª 2009 FEBS J.-W Seol et al Discussion TRAIL is a good candidate for cancer therapy as it selectively induces apoptosis in tumour cells, with little or no effect on normal cells [25] It has recently been reported that rheumatoid arthritis synovial tissue and fibroblasts both express high levels of DR5 (TRAILR2), are highly susceptible to DR5-mediated apoptosis, and DR5 may be a selective marker for rheumatoid arthritis [10] In addition, TRAIL protein is produced in rat arthritic cartilage and plays an important role in the pathogenesis of OA [11] Our study showed that TRAIL and ubiquitin protein expression in the synovial fluid from osteoarthritic joints was increased compared with that in control joints Changes in TRAIL and ubiquitin levels may be linked to the progression of inflammation and may be detected in the synovial fluid These changes could be associated with a natural history of OA and may be beneficial in the detection of patients at risk of rapidly progressing disease Articular cartilage is an avascular tissue that functions at an oxygen tension lower than that of most other tissues Articular cartilage derives both its nutrition and oxygen supply through diffusion from the synovial fluid and the subchondral bone [19,20,26] It has been reported that the partial pressure of oxygen in the synovial fluid of joints affected by OA is between 40 and 85 mmHg, corresponding to an oxygen concentration of approximately 6–11% [27] It has been estimated that articular chondrocytes in the deepest layers have access to no more than 1–6% O2 [20,22] Moreover, mitochondria are sparse in the articular chondrocytes, occupying only 1–2% of the intracellular volume [28], compared with 15–20% in other typical animal cells (for example, the liver) Marcus [29] and Otte [30] observed that chondrocytes produced ATP mostly through substrate-level phosphorylation during glycolysis However, oxygen tensions below 1% inhibit both glucose uptake and lactate production, as well as cellular RNA synthesis [29,30] This indicates that chondrocytes need at least some oxygen for their basal metabolic activity Therefore, hypoxia is considered to be a key factor in the growth and survival of chondrocytes Hypoxia is known to regulate the expression of many genes, but little is known about its role in either apoptosis or anti-apoptosis, especially in canine chondrocytes In this article, we investigated the possible effects of proteasome inhibition on TRAIL-induced apoptosis under normoxic and hypoxic conditions We found that TRAIL and ALLN alone did not induce apoptosis, but combined treatment of the cells with ALLN and TRAIL increased apoptosis markedly Hypoxic condition inhibits articular chondrocyte death to 60% in canine chondrocytes However, ALLN ⁄ TRAIL cotreatment-induced apoptosis of canine chondrocytes was inhibited significantly under hypoxic conditions This suggests that hypoxia can inhibit apoptotic activity in canine chondrocytes, and may therefore suppress the development and progression of OA Cell death in osteoarthritic cartilage possesses certain features of apoptosis or programmed cell death [31] Apoptosis is mediated by a cascade of aspartatespecific cysteine proteases or caspases, and increased caspase expression has been correlated with reduced cell density in human osteoarthritic cartilage [32] The present study demonstrated that ALLN pretreatment with TRAIL increased the activation of caspase-8 under normoxic conditions, but that caspase-8 activation was inhibited under hypoxic conditions In addition, the enhancing effect of proteasome inhibition on TRAIL-induced apoptosis was completely inhibited by the pan-caspase inhibitor, z-VAD-fmk Taken together, our data indicated that proteasome inhibition enhanced TRAIL-induced cell death via the caspase pathway, the key regulator of the TRAIL-induced cell death pathway in canine chondrocytes Furthermore, cotreatment of cells with both ALLN and TRAIL increased JNK phosphorylation under normoxic conditions, but this increase was inhibited under hypoxic conditions This suggests that the protective role of hypoxia involves the inhibition of caspase activation and JNK phosphorylation Mitochondria are central regulators of apoptosis [33,34] and may also be involved in chondrocyte death during bone development The activities of respiratory chain complexes II and III and the mitochondrial membrane potential are significantly reduced in cultured human chondrocytes from osteoarthritic donors when compared with normal donors [35] In this study, we demonstrated that hypoxic conditions prevented ROS generation and restored the loss of MTP seen after cotreatment with ALLN and TRAIL These findings suggest that the mitochondrial respiratory chain complexes are probable sites of ROS production, and that the inhibition of depolarization of MTP during hypoxia probably induces a decrease in ROS levels in canine chondrocytes In conclusion, the present study has demonstrated that proteasome activity, ubiquitinated protein, TRAIL and ROS are altered significantly in synovial fluid acquired from experimentally induced osteoarthritic joints At the cellular level, proteasome inhibition markedly enhances TRAIL-induced apoptosis through the activation of caspase-8, the phosphorylation of JNK protein, a decrease in MTP and the gener- FEBS Journal 276 (2009) 7375–7385 ª 2009 The Authors Journal compilation ª 2009 FEBS 7381 Hypoxic condition inhibits articular chondrocyte death J.-W Seol et al ation of ROS in primary cultured canine chondrocytes However, the enhanced apoptosis of chondrocytes induced by this combined treatment is inhibited under hypoxic conditions All these findings suggest that proteasome inhibition and TRAIL play a pivotal role in canine chondrocyte death and cartilage degradation These findings indicate that the maintenance of hypoxic conditions in cartilage inhibits articular chondrocyte apoptosis and may suppress the progression of arthritis Materials and methods Induction of OA Beagle dogs (n = 20) with a mean ± SD age of 1.4 ± 0.4 years and a mean ± SD weight of 10.2 ± 1.4 kg were used A right stifle joint medial arthrotomy was performed The cranial cruciate and the medial collateral ligaments were transected and a medial meniscectomy was performed The experimental animals were given intravenous crystalloid fluids (10 mLỈkg)1Ỉh)1) The surgical area was shaved and prophylactic antibiotic, cephalexin (Methilexin InjÒ; Union Korea Pharm Co Ltd., Seoul, South Korea), 25 mgỈkg)1 intravenously, was administered h before surgery The experimental animals were premedicated with atropine sulfate (Atropin Sulfate InjÒ; Dai Han Pharm Co Ltd., Seoul, South Korea), 0.05 mgỈkg)1, subcutaneously Anaesthesia was induced with propofol (Anepol InjÒ; Hana Pharm Co Ltd., Seoul, South Korea), mgỈkg)1 intravenously, and maintained with enflurane and oxygen During surgery, the jaw reflex, ocular reflex, heart rate (using electrocardiogram) and respiratory rate (using capnography) were monitored Based on these data, we changed the vaporizer settings if the experimental animals were in deep or light anaesthesia After surgery, postoperative treatment was given with butophanol (Butopan InjÒ; Hana Pharm Co Ltd.), 10 mgỈkg)1 intramuscularly, every 12 h for days for pain relief After days, no analgesic drug was given as the progress of OA was graded using a clinical scoring system, such as lameness, joint mobility and weight bearing All procedures employed in the animal experiments were approved by the Standard Operation Procedure of the Institutional Animal Care and Use Committee, Jeonju, South Korea Synovial fluid preparation Synovial fluid was collected 12 weeks after the induction of OA Briefly, experimental animals were sedated with acepromazine (Sedazect Inj; Samwoo Pharm Co Ltd., Seoul, South Korea), 0.2 mgỈkg)1 intravenously, and placed in ventrodorsal recumbency with the right stifle joints flexed Digital pressure was applied to the medial side of the straight patellar ligament A 21-gauge spinal needle was inserted through the fat pad into the intercondylar space lateral to the straight patellar ligament Chondrocyte isolation Normal canine knee cartilage was obtained from the knee joints of beagles (2-year-old females) The cartilage surfaces were first rinsed with sterile NaCl ⁄ Pi The cartilage slices were chopped and incubated with 0.25% trypsin for 30 min, followed by 0.1% collagenase (Sigma-Aldrich, St Louis, MO, USA; #C6885) treatment for h in Dulbecco’s modified Eagle’s medium (Invitrogen-Gibco, Grand Island, NY, USA) supplemented with 10% (v ⁄ v) fetal bovine serum (Invitrogen-Gibco) and antibiotics (100 lgỈmL)1 gentamycin and 100 lgỈmL)1 penicillin–streptomycin) Cells were filtered through a 70 lm cell strainer (Falcon, Franklin Lakes, NJ, USA), washed twice with NaCl ⁄ Pi and then seeded into tissue culture flasks The total cell number was calculated using a haemocytometer Cell viability test Evaluation of OA Experimental animals were sacrificed at 12 weeks to evaluate the severity of OA after surgery Levels of macroscopic synovial inflammation and cartilage damage were evaluated with digital high-resolution photographs The severity of synovial inflammation was graded on the basis of colour, angiogenesis and fibrillation: grade 0, no inflammation; 7382 grade 1, slight inflammation; grade 2, strong inflammation The cartilage damage severity of the femoral condyles and tibial plateau was graded from to 4: grade 0, smooth surface; grade 1, slight fibrillation; grade 2, fibrillation with shallow grooves; grade 3, deep and sharp grooves; grade 4, deep and sharp grooves with surrounding damage Radiographic examinations were also performed The severity of osteophyte formation in the femoropatellar, lateral femorotibial, medial femorotibial and central femorotibial joints was graded from to 3: grade 0, absent; grade 1, mild; grade 2, moderate; grade 3, severe The degree of synovial effusion was graded from to 3: grade 0, absent; grade 1, mild; grade 2, moderate; grade 3, severe Two independent observers assigned individual scores, and all values were averaged and used in the statistical analyses Canine chondrocytes were adjusted to 1.0 · 106 cells per well in 12-well plates, pretreated with ALLN (Sigma, St Louis, MO, USA) for 12 h, and then further incubated with recombinant TRAIL protein for 12 h under normoxic (21% O2) and hypoxic (1% O2) conditions at the indicated doses Cellular morphology was photographed under light FEBS Journal 276 (2009) 7375–7385 ª 2009 The Authors Journal compilation ª 2009 FEBS J.-W Seol et al microscopy (Nikon, Tokyo, Japan), and cell viability was determined using the crystal violet staining method, as described previously [36] Briefly, the cells were stained for 10 at room temperature with staining solution (0.5% crystal violet in 30% ethanol and 3% formaldehyde), washed four times with water and then dried The cells were then lysed with 1% SDS solution and the absorbance was measured at 550 nm The cell viability was calculated based on the relative dye intensity compared with controls Western blot assay To prepare whole-cell lysates, cells were harvested and resuspended in lysis buffer (25 mm Hepes, pH 7.4, 100 mm NaCl, mm EDTA, mm MgCl2, 0.1 mm dithiothreitol and protease inhibitor mixture) Synovial fluid was diluted 10 times with NaCl ⁄ Pi Proteins were electrophoretically resolved on an 8–15% SDS gel, and western blots were performed as described previously [37] Equal amounts of the lysate protein were also resolved on an 8–15% SDS-PAGE gel and then electrophoretically transferred to a nitrocellulose membrane The immunoreactivity was detected through sequential incubation with horseradish peroxidaseconjugated secondary antibodies and ECL reagents (Amersham corp., Burlington, MA, USA) The antibodies used for western blotting analyses were caspase-8 (AAP-118) (Stressgen, Victoria, Canada), Ubiquitin (Cell Signaling Technology, Danvers, MA, USA), TRAIL (Santa Cruz Biotechnology, Santa Cruz, CA, USA; sc-8440), c-Jun N-terminal kinase–stress-activated protein kinase (JNK) and the phosphorylated form (p-JNK) (Upstate Biotechnology, Lake Placid, NY, USA) Proteasome activity test Proteasome activity was measured as described previously [38] The cells were collected by centrifugation and the synovial fluid was diluted 10 times with NaCl ⁄ Pi Protein concentrations of synovial fluid and cytoplasm were determined using the Bradford protein assay kit (Bio-Rad, Hercules, CA, USA) Two hundred micrograms of synovial fluid protein and cytoplasm protein were added to the assay buffer (20 mm Tris ⁄ HCl, pH 8.0, mm ATP, mm MgCl2) in the presence of the synthetic fluorogenic substrate SucLLVY-AMC to a final concentration of 60 lm (SigmaAldrich) suspended in a final volume of mL The tubes were incubated at 30 °C for 30 min, after which the reaction was terminated through the addition of mL of cold ethanol The lysate was spun at 12 000 g for 10 at °C Fluorescence was measured at 380 nm excitation and 440 nm emission using a fluorescence plate reader (SpectraMax fluorometer with the softmax program; Molecular Probes, Eugene, OR, USA) Hypoxic condition inhibits articular chondrocyte death Evaluation of MTP The level of MTP was determined using a lipophilic cation, JC-1 (Molecular Probes) Briefly, chondrocytes were isolated from cartilage obtained from osteoarthritic joints The cells were collected by centrifugation, washed twice with NaCl ⁄ Pi and resuspended in 500 lL of NaCl ⁄ Pi containing JC-1 at a concentration of 10 lm After 30 of incubation at 37 °C, the cells were photographed using a microscope (ECLIPSE 80 i, Nikon), and red fluorescence was monitored with a fluorescence plate reader (SpectraMax fluorometer with the softmax program; Molecular Probes), with 490 and 590 nm as excitation and emission wavelengths, respectively Determination of ROS ROS levels, particularly the levels of intracellular hydroperoxides, were assessed using the oxidant-sensitive dye 2¢,7¢dichlorodihydrofluorescein diacetate (DCFH2-DA) The cells treated with ALLN and TRAIL for 12–24 h were washed twice with NaCl ⁄ Pi and incubated with 10 lm DCFH2-DA in sodium pyruvate containing Dulbecco’s modified Eagle’s medium for h at 37 °C After DCFH2DA incubation, the cells were washed and further incubated in sodium-containing medium for 10 to allow de-esterification to occur The cells were then collected, and the fluorescence signals corresponding to intracellular ROS were monitored at 490 nm excitation and 525 nm emission using a fluorescence plate reader (SpectraMax fluorometer with the softmax program, Molecular Probes) Hypoxic conditions A sealed chamber was used to culture the chondrocytes at low oxygen tension (1%) A gas mixture of 1% O2, 5% CO2 and 94% N2 was added to the sealed chamber, and ambient air was evacuated through an outlet tube The oxygen flow was allowed to stream through the chamber for 2–3 to maintain the desired oxygen tension inside the chamber Culture plates were incubated in sealed chambers containing 1% O2 at 37 °C For the normoxic condition (21% O2 tension), the chondrocytes were incubated at 37 °C in a 95% humidified atmosphere with 5% CO2 There were two controls (normoxia and hypoxia) in this experiment The hypoxia control was handled in the same type of sealed unit as used for 1% O2 Statistical evaluation All data are expressed as the mean ± SD, and were compared using Student’s t-test and the ANOVA Duncan test with the sas statistical package The results were considered to be significant at P < 0.05 and P < 0.01 FEBS Journal 276 (2009) 7375–7385 ª 2009 The Authors Journal compilation ª 2009 FEBS 7383 Hypoxic condition inhibits articular chondrocyte death J.-W Seol et al Acknowledgement This work was supported by a Korea Research Foundation Grant from the Regional Research Universities Program ⁄ Center for Healthcare Technology Development References 12 Hashimoto S, Ochs RL, Komiya S & Lotz M (1998) Linkage of 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TRAIL-induced apoptosis by CCCP Oncol Rep 18, 71–76 37 Park SY, Billiar TR & Seol DW (2002) Hypoxia inhibition of apoptosis induced by tumor necrosis factorrelated apoptosis-inducing ligand (TRAIL) Biochem Biophys Res Commun 291, 150–153 38 Jamaluddin M, Casola A, Garofalo RP, Han Y, Elliott T, Ogra PL & Brasier AR (1998) The major component of IkappaBalpha proteolysis occurs independently of the proteasome pathway in respiratory syncytial virusinfected pulmonary epithelial cells J Virol 72, 4849–4857 FEBS Journal 276 (2009) 7375–7385 ª 2009 The Authors Journal compilation ª 2009 FEBS 7385 ... examination of the articular cartilage after experimentally induced OA Articular cartilage of the femoral condyles from the experimental joints was examined to assess any macroscopic damage caused... Evaluation of articular cartilage after experimentally induced osteoarthritis (A) Photomicrographs of articular cartilage (B) The evaluation of osteoarthritis in the right and left joints of dogs... intercondylar space lateral to the straight patellar ligament Chondrocyte isolation Normal canine knee cartilage was obtained from the knee joints of beagles (2-year-old females) The cartilage surfaces