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DcR3 induces cell proliferation through MAPK signaling in chondrocytes of osteoarthritis
Osteoarthritis and Cartilage 19 (2011) 903e910
DcR3 induces cell proliferation through MAPK signaling in chondrocytes of osteoarthritis S. Hayashi, T. Nishiyama*, Y. Miura, T. Fujishiro, N. Kanzaki, S. Hashimoto, T. Matsumoto, M. Kurosaka, R. Kuroda Department of Orthopaedic Surgery, Kobe University, Graduate School of Medicine, Kobe, Japan
a r t i c l e i n f o
s u m m a r y
Article history: Received 21 January 2011 Accepted 11 March 2011
Introduction: Decoy receptor 3 (DcR3), a soluble receptor belonging to the tumor necrosis factor (TNF) receptor superfamily, competitively binds and inhibits the TNF family including Fas-ligand (Fas-L), lymphotoxin-like inducible protein that competes with glycoprotein D for binding herpesvirus entry mediator on T-cells (LIGHT) and TNF-like ligand 1A (TL1A). In this study, we investigated the functions of DcR3 on osteoarthritis (OA) chondrocytes. Methods: Expressions of DcR3 in chondrocytes were measured by realtime Reverse Transcriptase-Polymerase Chain Reaction (RT-PCR). Expression of DcR3 in sera and joint fluids was measured by enzymelinked immunosorbent assay (ELISA). Chondrocytes were incubated with DcR3-Fc chimera protein (DcR3-Fc) before induction of apoptosis by Fas-L and apoptosis was detected with terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick end labelling labeling (TUNEL) staining and Western blotting of caspase 8 and poly (ADP-ribose) polymerase (PARP). Chondrocytes were incubated with DcR3-Fc and the proliferation was analyzed by 4-[3-(4-iodophenyl)-2-(4-nitrophenyl)-2H-5-tetrazolio]1,3-benzene disulfonate (WST) assay. Phosphorylation of Extracellular Signal-Regulated Kinase (ERK), P38 mitogen-activated protein kinase (MAPK) and Jun N-terminal Kinase (JNK) in chondrocytes was measured by Western blotting after incubation with DcR3-Fc, Mitogen-activated protein kinase kinase (MEK1/2) inhibitor, or P38 MAPK inhibitor. Chondrocytes were treated with DcR3Fc after pre-incubation with blocking antibody of Fas-L, LIGHT and TL1A, and proliferation or phosphorylation of ERK was analyzed. Results: DcR3 was expressed in OA and normal chondrocytes. DcR3-Fc protects chondrocytes from Fasinduced apoptosis. DcR3-Fc increased chondrocytes proliferation and induced the phosphorylation of ERK specifically. DcR3-induced chondrocytes proliferation was inhibited by pre-incubation of PD098059 or blocking Fas-L antibody. DcR3 increased chondrocytes proliferation in OA chondrocytes, but did not in normal. Conclusion: DcR3 regulates the proliferation of OA chondrocytes via ERK signaling and Fas-induced apoptosis. Ó 2011 Osteoarthritis Research Society International. Published by Elsevier Ltd. All rights reserved.
Keywords: Decoy receptor 3 Chondrocyte Apoptosis ERK Fas-ligand Proliferation
Introduction Decoy receptor 3 (DcR3)/TR6/M68 is a member of the tumor necrosis factor receptor (TNFR) superfamily identified1. DcR3 lacks the transmembrane domain of conventional TNFRs and is believed to be a secreted protein1. DcR3 is expressed in some normal tissues
* Address correspondence and reprint requests to: T. Nishiyama, Department of Orthopaedic Surgery, Kobe University, Graduate School of Medicine, 7-5-1 Kusunoki-cho, Chuo-ku, Kobe 650-0017, Japan. Tel: 81-78-382-5985; Fax: 81-78-351-6944. E-mail address: [email protected] (T. Nishiyama).
including colon, stomach, spleen, lymph node, spinal cord, pancreas, and lung1,2. DcR3 is overexpressed in tumor cells including lung and colon cancers1, gliomas, and gastrointestinal tract tumors2. Overexpression of DcR3 might benefit tumors by helping them to avoid the cytotoxic and regulatory effects of Fasligand (Fas-L)1, lymphotoxin-like inducible protein that competes with glycoprotein D for binding herpesvirus entry mediator on T-cells (LIGHT)3, and TNF-like ligand 1A (TL1A)4. Furthermore, DcR3 is expressed in synovial cells of both osteoarthritis (OA) and rheumatoid arthritis (RA)5. Overexpressed DcR3 in rheumatoid synovial cells stimulated with proinflammatory tumor necrosis factor (TNF) alpha protects the cells from apoptosis5,6.
1063-4584/$ e see front matter Ó 2011 Osteoarthritis Research Society International. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.joca.2011.03.005
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S. Hayashi et al. / Osteoarthritis and Cartilage 19 (2011) 903e910
OA, the most common degenerative disease of human articular cartilage, is characterized by extracellular matrix damage and an important loss in tissue cellularity7. Cartilage cells are mainly responsible for the anabolicecatabolic balance required for matrix maintenance and tissue function8. OA cartilage has a higher proportion of apoptotic chondrocytes than normal cartilage9 and apoptosis may play a role in diseases involving articular cartilage degeneration10. Therefore, DcR3 might be involved in the regulation of chondrocyte apoptosis. Further, recently, DcR3 was reported to function as a ligand. The expression of Human Leukocyte Antigen DR-1 (HLA-DR) and other costimulatory molecules such as CD40, CD54/ICAM-1, CD1a and CD80/B7.1 was down-regulated by DcR3-Fc and the costimulatory molecule CD86/B7.2 was up-regulated in DCs11. Hence, we speculated that DcR3 might be expressed in chondrocytes and regulate apoptosis and proliferation of chondrocytes possibly resulting in cartilage regeneration. In this study, therefore, we investigated DcR3 expression in chondrocytes and analyzed the function of DcR3 in apoptosis and proliferation, and suggested that DcR3 plays a substantial role in the physiology of OA chondrocytes. Materials and methods Preparation of cartilage Cartilage tissues were obtained during total hip joint replacement surgery from 19 patients with OA. Diagnosis of OA was based on clinical, laboratory, and radiographic evaluations. Normal cartilage tissues were obtained during surgery of femoral neck fracture from seven patients with no history of joint disease and with macroscopically normal cartilage. All the samples were obtained in accordance with the World Medical Association Declaration of Helsinki Ethical Principles for Medical Research Involving Human Subjects. Cell culture Chondrocytes were isolated and cultured from cartilage tissues. Tissues were minced and incubated with trypsin (0.5 mg/ml) (Sigma, St. Louis, MO, USA) for 15 min at 37 C, after which the cartilage was treated with Dulbecco’s modified Eagle’s medium (DMEM; Gibco BR, Grand Island, NY, USA) containing 0.2% of collagenase (Sigma) at 37 C for 15 h. Dissociated cells were cultured in DMEM supplemented with 10% fetal bovine serum (FBS; Biowhittaker, Walkersville, ML, USA) and 100 U/ml of penicillinestreptomycin. After overnight culture, non-adherent cells were removed, and adherent cells were further incubated in fresh medium. All experiments were conducted using first passage cells. Quantification of DcR3, Fas-L and Fas mRNA expression Chondrocytes were cultured in 6-well plates with various stimulations, and RNA was extracted with QIAshredder and RNeasy Mini kit (Qiagen, Hilden, Germany) according to the manufacturer’s protocol. 1 mg of total RNA was reverse-transcribed to first-strand cDNA with 1.25 mM oligo dT primer in 40 ml PCR buffer II containing 2.5 mM MgC12, 0.5 mM dNTP mix, 0.5 U RNase inhibitor, and 1.25 U MuLV reverse transcriptase (Perkin Elmer, Foster City, CA, USA) at 42 C for 60 min. The relative expression levels of mRNA encoding DcR3, Fas-L or Fas in chondrocytes were analyzed by TaqManÒ realtime PCR using an ABI prism 7500 sequence detection system (Applied Biosystems, Foster City, CA, USA). Pre-designed primers and probes for human DcR3, Fas-L, Fas and human glyceraldehyde-3-phosphate
dehydrogenase (GAPDH) to be used as the control were obtained from Applied Biosystems. Quantification of DcR3 protein concentration in sera or joint fluid In order to measure DcR3 in serum and joint fluid, we used the human DcR3 enzyme-linked immunosorbent assay (ELISA) kit (Bender Med Systems, Vienna, Austria) according to the manufacturer’s protocol. Briefly, 100 ml of each sample were incubated in duplicate in microplates coated with anti-DcR3 monoclonal antibody for 2 h. Following incubation, biotin-conjugate anti-DcR3 monoclonal antibody was added and incubated as a primary antibody for 2 h. After the microplate had been washed, streptavidinhorseradish peroxidase (HRP) was added and incubated for 1 h. After washing off any unbound streptavidin-HRP, tetramethylbenzidine was added as a substrate and the absorbance was measured at 450 nm after 20 min in a microplate reader (BioTek Instruments, Burlington, VT, USA). The absorbance of each sample was plotted against a standard curve produced by serial dilutions of recombinant human DcR3-Fc in duplicate. The concentrations of DcR3 in serum and joint fluid were calculated by logarithmic analysis. All samples with an absorbance of less than zero according to the standard curve were discarded from the final analysis. Fas-induced apoptosis in chondrocytes Before the induction of apoptosis, chondrocytes were pretreated with 0.1, 1, 10 and 100 ng/ml of recombinant human DcR3Fc chimera protein (DcR3-Fc) (R&D Systems) for 24 h in Opti Minimum essential medium (MEM) (Invitrogen, Carlsbad, CA, USA). DcR3-Fc and recombinant DcR3 protein (rDcR3) have the same binding affinity to LIGHT and the same inhibitory effect on the development of Cytotoxic T-lymphocyte (CTL) in mice indicating that the biological effect of DcR3-Fc is equivalent to rDcR312. Fas-induced apoptosis was introduced by incubation with 100 ng/ml of recombinant human Fas-ligand (hFas-L) (R&D Systems) for an additional 12 h. Fas-induced apoptotic signals were confirmed by terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick end labeling (TUNEL) staining and the detection of cleavage of caspase 813 and poly (ADP-ribose) polymerase (PARP)9. Proliferation assay Chondrocytes were seeded in 96-well plates at a density of 5 103 cells per well in DMEM medium supplemented with 10% FBS. After plating cells for overnight, chondrocytes were treated with DcR3-Fc or IgG-Fc (Millipore, Billerica, MA, USA) as control to eliminate the effects of Fc. In some cases, chondrocytes were preincubated with blocking antibody of Fas-L (BD Pharmingen, San Jose, CA, USA), LIGHT and TL1A (Biolegend, San Diego, CA, USA) or IgG (Acris, Littleton, CO, USA) as control for 12 h. Cell proliferation was assessed using Cell-Counting Kit-8 (Dojindo, Kumamoto, Japan) (4-[3-(4-iodophenyl)-2-(4-nitrophenyl)-2H-5-tetrazolio]1,3-benzene disulfonate (WST) assay) according to the manufacturer’s instruction. The principle of WST assay is following. Tetrazolium salt-8 (WST-8) is cleaved to soluble formazan dye by the succinate-tetrazolium reductase, which exists in mitochondrial respiratory chain and is active only in viable cells. Total activity of this mitochondrial succinate-tetrazolium reductase in a sample rises with the increase of viable cells. The absorbance of formazan dye solution is in direct proportion to the number of viable cells. Therefore, this assay is designed for non-radioactive quantification of cell growth and viability in proliferation. The values, corresponding to the number of viable cells, were read at OD 450 nm with Microplate Reader (BioTek Instrument, Winooski, VT, USA).
S. Hayashi et al. / Osteoarthritis and Cartilage 19 (2011) 903e910
DcR3-Fc and rDcR3 have the same binding affinity to LIGHT and the same inhibitory effect on the development of CTL in mice indicating that the biological effect of DcR3-Fc is equivalent to rDcR312. Western blotting Chondrocytes were washed three times with Phosphate Buffered Saline (PBS) and lysed in hypotonic lysis buffer (25 mM Tris, 1% NP-40, 150 mM NaCl, 1.5 mM Ethylene glycol tetraacetic acid (EGTA)) supplemented with protease and phosphatase inhibitor mix (Roche Diagnostics, Basel, Switzerland) on ice for 20 min. The lysates were centrifuged at 15,000 rpm for 20 min to remove cellular debris and the supernatants were collected. Cytoplasmic proteins were quantified by the Bradford method with protein assay reagent (BioRad, Hercules, CA, USA), and diluted to an equal concentration with hypotonic buffer. Each sample was mixed with 3 electrophoresis sample buffer (BioRad) and electrophoresed on 7.5e15% polyacrylamide gradient gel (Biocraft, Tokyo, Japan) and transblotted electrically onto the blotting membrane (GE Healthcare, Connecticut, CT, USA). The expression of DcR3 protein was detected using rabbit antihuman DcR3 polyclonal Ab (R&D systems). The expression of caspase 8 was detected using mouse anti-human caspase 8 monoclonal Ab (Cell Signaling Technology, Beverly, MA, USA). The expression of PARP was detected using rabbit anti-human PARP polyclonal Ab (Cell Signaling Technology). The expression of phospho-Extracellular Signal-Regulated Kinase (ERK) or ERK was detected using mouse anti-human phospho-ERK Ab (Cell Signaling Technology) or rabbit anti-human ERK Ab (Cell Signaling Technology). The
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expression of phospho-P38 mitogen-activated protein kinase (MAPK) or P38 MAPK was detected using rabbit anti-human phospho-P38 MAPK Ab (Cell Signaling Technology) or rabbit anti-human P38 MAPK Ab (Cell Signaling Technology). The expression of phospho-Jun N-terminal Kinase (JNK) or JNK was detected using rabbit anti-human phospho-JNK Ab (Cell Signaling Technology) or rabbit anti-human JNK Ab (Cell Signaling Technology). HRP conjugated goat anti-rabbit IgG Ab (GE healthcare) or HRP conjugated rabbit anti-mouse IgG Ab (GE healthcare) was used for a secondary antibody, and the signals were visualized by ECL plus reagent (GE healthcare) with Chemilumino analyzer LAS-3000 mini (FUJI FILM, Tokyo, Japan). The expression of proteins was determined by semi-quantification of digitally captured image using the public domain NIH Image program (developed at the U.S. National Institutes of Health and available on the Internet at http://rsb.info.nih.gov/nihimage/). Values were normalized to alpha-tublin expression. TUNEL staining 2 105 chondrocytes were cultured in 8-well chamber slides (Nunc Roskilde, Denmark). After various stimulations, the chondrocytes were fixed with 4% neutral buffered formalin for 10 min and apoptotic cells were determined using TUNEL assay kit (Wako) according to the manufacturer’s protocol. Statistical analysis Statistical analysis was performed using one-way analysis of variance, with Tukey’s post hoc test for multiple comparisons of paired samples. Results are presented as mean values with 95%
Fig. 1. (a) Expression levels of DcR3, Fas-L or Fas mRNA were quantified by realtime PCR. Expression ratios of DcR3, Fas or Fas-L (OA chondrocytes/Normal chondrocytes) are shown. Columns represent mean values with 95% CI. The results shown are the average of seven individual samples. Values were normalized to GAPDH expression. (b) Human Serum or joint fluid levels of DcR3 protein were measured by ELISA. Control sera was collected from 10 healthy subjects, OA sera and joint fluid were from 10 subjects with OA. Columns represent mean values with 95% CI.
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Fig. 2. (a and b) Effect of hFas-L and DcR3-Fc protein for Fas-induced apoptosis of chondrocytes. (a) Columns represent mean values with 95% CI. Columns represent of TUNEL positive cells, at least 300 cells were counted by a blinded observer in the same experiment. The results shown are the average of four individual samples. (b) Expressions of cleaved caspase 8 and PARP were analyzed by Western blotting. The results shown represent three independent experiments.
confidence intervals (95% CI), and were considered statistically significant for P < 0.05. Results
between OA (mean ¼ 0.924, 95% CI ¼ 0.56e1.29, N ¼ 7) and normal chondrocytes (mean ¼ 1, 95% CI ¼ 0.78e1.22, N ¼ 7). However, the levels of Fas-L were significantly higher in OA (mean ¼ 2.97, 95% CI ¼ 2.14e3.79, N ¼ 7) than in normal chondrocytes (mean ¼ 1, 95% CI ¼ 0.76e1.24, N ¼ 7) (Fig. 1a).
Expression levels of DcR3 mRNA were not changed between OA and normal chondrocytes
DcR3 is expressed in joint fluids of patients with OA
In order to compare the expression levels of DcR3, Fas-L or Fas mRNA between OA and normal chondrocytes, we analyzed mRNA levels by realtime Reverse Transcriptase-Polymerase Chain Reaction (RT-PCR). The expression levels of DcR3 were not significantly changed between OA (mean ¼ 0.89, 95% CI ¼ 0.50e1.28, N ¼ 7) and normal chondrocytes (mean ¼ 1, 95% CI ¼ 0.78e1.22, N ¼ 7). The expression levels of Fas were not significantly changed
In order to analyze the expression of DcR3 protein in sera and joint fluid, we quantified the concentration of DcR3 by DcR3 ELISA kit. The average concentration of serum DcR3 was 0.33 ng/ml in OA patients (95% CI ¼ 0.04e0.62, N ¼ 10), and 0.24 ng/ml in healthy donor (95% CI ¼ 0.03e0.45, N ¼ 10) (Fig. 1b). There was no significant difference between OA and healthy subjects. While, the average concentration of DcR3 in joint fluid of OA patients was
Fig. 3. (a) Effect of DcR3 on chondrocyte proliferation analyzed by WST assay. Chondrocytes were incubated with 5 mg/ml of IgG-Fc or 1, 5 mg/ml of DcR3-Fc for 48 h. Columns show the ratio of OD 450 nm value in comparison with untreated chondrocytes. The results shown are the average of four individual samples. Columns represent mean values with 95% CI. (b) Effect of DcR3-Fc for ERK, P38 MAPK, or JNK phosphorylation analyzed by Western blotting. Chondrocytes were incubated with 5 mg/ml of DcR3-Fc or 5 mg/ml of IgG-Fc for 0, 5, 10 and 30 min. The results shown represent three independent experiments.
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Fig. 4. Effects of PD098059 (PD) and SB203580 (SB) against DcR3-induced proliferation of chondrocytes. (a) Chondrocyte proliferation was analyzed by WST assay. Chondrocytes were incubated with 5 mg/ml of IgG-Fc, 5 mg/ml of DcR3-Fc, 50 mM of PD098059 (R&D) or 10 mM of SB203580 (R&D) for 48 h. In some cases, chondrocytes were pre-treated with 50 mM of PD098059 or 10 mM of SB203580 for 30 min, and incubated with 5 mg/ml of DcR3-Fc for 48 h. Columns show the ratio of OD 450 nm value in comparison with untreated chondrocytes. The results shown are the average of three individual samples. Columns represent mean values with 95% CI. (b) Phosphorylation of ERK was analyzed by Western blotting. Chondrocytes were incubated with 5 mg/ml of IgG-Fc, 5 mg/ml of DcR3-Fc, 50 mM of PD098059 or 10 mM of SB203580 for 10 min. In some cases, chondrocytes were pretreated with 50 mM of PD098059 or 10 mM of SB203580 for 30 min, and incubated with 5 mg/ml of DcR3-Fc for 10 min. The results shown represent three independent experiments.
Fig. 5. Effects of blocking antibody of Fas-L, LIGHT and TL1A for DcR3-induced chondrocytes proliferation. (a) Chondrocyte proliferation was analyzed by WST assay. Chondrocytes were pre-incubated with 100 ng/ml of blocking antibody of Fas-L, 5 mg/ml of blocking antibody of LIGHT and TL1A or 5 mg/ml of IgG as control for 12 h. After washing cells with PBS, chondrocytes were incubated with 5 mg/ml of DcR3-Fc for 48 h. Columns show the ratio of OD 450 nm value in comparison with 5 mg/ml of IgG pre-treatment. The results shown are the average of four individual samples. Columns represent mean values with 95% CI. (b) Phosphorylation of ERK was analyzed by Western blotting. After pre-incubation with IgG, blocking antibody of Fas-L, LIGHT and TL1A for 12 h, chondrocytes were washed with PBS and incubated with 5 mg/ml of DcR3-Fc for 0, 5, 10 and 30 min.
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1.44 ng/ml (95% CI ¼ 0.90e2.00, N ¼ 10). The average concentration of DcR3 in joint fluid of OA patients was much higher than in sera (Fig. 1b). DcR3 protein inhibited Fas-induced apoptosis in chondrocytes TUNEL positive apoptotic cells induced by Fas-L were significantly decreased in a dose dependent manner when chondrocytes were pre-incubated with DcR3-Fc protein (Untreated: mean ¼ 8, 95% CI ¼ 5.7e10.3, N ¼ 4) (0 ng/ml DcR3Fc: mean ¼ 44.6, 95% CI ¼ 35.5e53.8, N ¼ 4) (0.1 ng/ml DcR3-Fc: mean ¼ 34.9, 95% CI ¼ 31.8e38.1, N ¼ 4) (1 ng/ml DcR3Fc: mean ¼ 30.2, 95% CI ¼ 28.0e32.4, N ¼ 4) (10 ng/ml DcR3-Fc: mean ¼ 24.3, 95% CI ¼ 17.8e30.7, N ¼ 4) (100 ng/ml DcR3Fc: mean ¼ 13.9, 95% CI ¼ 5.7e22.1, N ¼ 4) (Fig. 2a). The cleavage of caspase 8 and PARP was increased by 100 ng/ml of Fas-L treatment. However, the cleavage of caspase 8 and PARP with Fas-L treatment was inhibited by pre-treatment with 100 ng/ml of DcR3-Fc (Fig. 2b). DcR3 increased OA chondrocytes proliferation via ERK phosphorylation WST assay revealed that chondrocytes proliferation were increased by the treatment of DcR3-Fc in a dose dependent manner (Untreated: mean ¼ 1, 95% CI ¼ 0.94e1.07, N ¼ 4) (IgG-Fc: mean ¼ 1.04, 95% CI ¼ 0.86e1.22, N ¼ 4) (1 mg/ml DcR3Fc: mean ¼ 1.20, 95% CI ¼ 1.11e1.30, N ¼ 4) (5 mg/ml DcR3-Fc: mean ¼ 1.32, 95% CI ¼ 1.24e1.39, N ¼ 4) (Fig. 3a). In order to investigate the signal transduction of DcR3, we tested phosphorylation of ERK, P38 MAPK and JNK in chondrocytes with the treatment of 5 mg/ml of DcR3-Fc. Western blotting revealed that phosphorylation of ERK was increased to 12.63 folds of untreated samples at 10 min and 3.12 folds at 30 min by treatment with 5 mg/ml of DcR3-Fc (Fig. 3b). While, neither P38 MAPK nor JNK signaling was activated by treatment with 5 mg/ml of DcR3-Fc for up to 30 min (Fig. 3b). Suppression of ERK activity inhibited DcR3-induced chondrocytes proliferation WST assay revealed that the effect of DcR3-Fc for chondrocytes proliferation was inhibited by treatment with specific Mitogen-
activated protein kinase kinase (MEK1/2) inhibitor PD098059, but not by specific P38 MAPK inhibitor SB203580 (Untreated: mean ¼ 1, 95% CI ¼ 0.91e1.09, N ¼ 3) (IgG-Fc: mean ¼ 1.01, 95% CI ¼ 0.93e1.10, N ¼ 3) (PD: mean ¼ 0.96, 95% CI ¼ 0.90e1.02, N ¼ 3) (SB: mean ¼ 1.04, 95% CI ¼ 0.99e1.09, N ¼ 3) (DcR3-Fc: mean ¼ 1.36, 95% CI ¼ 1.16e1.60, N ¼ 3) (DcR3-Fc þ PD: mean ¼ 1.10, 95% CI ¼ 0.94e1.26, N ¼ 3) (DcR3-Fc þ SB: mean ¼ 1.31, 95% CI ¼ 1.22e1.41, N ¼ 3) (Fig. 4a). Western blotting confirmed that the activation of ERK signaling by treatment with 5 mg/ml DcR3-Fc was inhibited by PD098059, but not by SB203580 (Fig. 4b).
Anti-Fas-L antibody inhibited the DcR3-induced chondrocytes proliferation through ERK signaling In order to figure out the mechanism of DcR3-induced signal transduction in chondrocytes proliferation, we tested the effect of blocking antibody of Fas-L, LIGHT and TL1A for DcR3-induced proliferation. WST assay revealed that the effect of DcR3-Fc for chondrocytes proliferation was inhibited by pre-incubation with blocking antibody of Fas-L, but not by blocking antibody of LIGHT or TL1A (IgG: mean ¼ 1, 95% CI ¼ 0.98e1.02, N ¼ 4) (Fas-L: mean ¼ 0.61, 95% CI ¼ 0.56e0.67, N ¼ 4) (LIGHT: mean ¼ 1.03, 95% CI ¼ 0.71e1.36, N ¼ 4) (TL1A: mean ¼ 0.99, 95% CI ¼ 0.98e1.02, N ¼ 4) (Fig. 5a). Western blotting showed that phosphorylation of ERK by treatment with 5 mg/ml of DcR3-Fc was inhibited by preincubation with blocking antibody of Fas-L, but not with blocking antibody of LIGHT or TL1A (Fig. 5b).
DcR3 did not increase proliferation of normal chondrocytes In order to compare the difference between OA and normal chondrocytes in response to DcR3 treatment, we tested chondrocytes proliferation by treatment of 5 mg/ml of DcR3-Fc in OA and normal chondrocytes. WST assay revealed that DcR3-Fc increased chondrocytes proliferation in OA chondrocytes, but did not in normal (Untreated normal: mean ¼ 1, 95% CI ¼ 0.87e1.13, N ¼ 4) (Untreated OA: mean ¼ 1, 95% CI ¼ 0.82e1.18, N ¼ 4) (IgG-Fc normal: mean ¼ 0.95, 95% CI ¼ 0.88e1.02, N ¼ 4) (IgG-Fc OA: mean ¼ 1.03, 95% CI ¼ 0.94e1.12, N ¼ 4) (DcR3-Fc normal: mean ¼ 1.09, 95% CI ¼ 0.88e1.30, N ¼ 4) (DcR3-Fc OA: mean ¼ 1.31, 95% CI ¼ 1.27e1.34, N ¼ 4) (Fig. 6).
Fig. 6. Comparison of DcR3 effect on proliferation between OA and normal chondrocytes. Chondrocytes were incubated with 5 mg/ml of IgG-Fc or DcR3-Fc for 48 h. Chondrocytes proliferation was analyzed by WST assay. Columns show the ratio of OD 450 nm value in comparison with untreated chondrocytes. White panels: Normal chondrocytes. Black panels: OA chondrocytes. The results shown are the average of four individual samples. Columns represent mean values with 95% CI.
S. Hayashi et al. / Osteoarthritis and Cartilage 19 (2011) 903e910
Discussion A significant body of knowledge about the mechanism of chondrocytes apoptosis was reported. Many manuscripts reported that chondrocytes apoptosis were induced via mitochondrial pathway or cytokines like TNF alpha14,15. We also have shown that mechanical stress induced chondrocytes apoptosis via p53-mitochondrial pathway14. While, it has been reported that several stimuli including Fas-L16 induced apoptosis in chondrocytes and a significant amount of FasL was found in synovial fluids from patients with OA16. Expression of Fas-L was higher in aged cartilage than in mature cartilage, which correlated with the decrease in viable cell density in rabbit articular cartilage during aging17. We confirmed that the expression of Fas-L was increased in OA chondrocytes. These findings suggest that Fas/ Fas-L is an important molecule for the regulation of apoptosis of chondrocytes in OA cartilage. DcR3 is expressed in a variety of tissues and tumors1,2, and regulates cell proliferation, however, the expression of DcR3 in chondrocytes was not investigated previously. In this study, we demonstrated that DcR3 is expressed in normal and OA chondrocytes similarly, and the expression levels of DcR3 protein in the joint fluid were higher than in sera. Average concentration of DcR3 in joint fluid of OA patients was 1.44 ng/ml. While, we also demonstrated that 1 ng/ml of DcR3-Fc inhibited Fas-induced apoptosis in chondrocytes. These results suggest that DcR3 protein is secreted into the joint and actually protects chondrocytes from death via Fas-induced apoptosis in physiological circumstance. However, the amount of DcR3 may be insufficient to bind to the increased amount of Fas-L in OA cartilage16,17 and incapable to inhibit Fas-induced apoptosis. From the standpoint of chondrocyte proliferation, MAPK signaling is one of the essential mitogenic signaling in many kinds of cells and plays a key role in a variety of cellular responses including proliferation, differentiation and apoptosis of cells18,19. We recently demonstrated that DcR3 has functions as a ligand to inhibit apoptosis in THP-1 macrophages by increasing very late antigen 4 (VLA4) expression20. Several studies have reported that soluble DcR3 activates reverse signaling as a ligand, inducing osteoclast formation from monocytes/macrophages via ERK and p38 MAPK signaling21, inducing actin reorganization and enhancing adhesion of monocytes22, and inducing dendritic cell apoptosis by activating Protein Kinase C (PKC-d) and JNK23,24. In this study we demonstrated that DcR3-Fc increased proliferation of chondrocytes through activation of ERK signaling pathway specifically, but not in p38 MAPK or JNK. DcR3-Fc fusion protein has been shown to have similar binding affinity and specificity as DcR325,26, and in our experiments control IgG-Fc had no effect on chondrocytes. Our results therefore are the first to suggest that DcR3 may regulate chondrocytes proliferation by binding Fas-L through ERK signaling. We further demonstrated that chondrocytes proliferation by DcR3-Fc treatment was different between OA and normal chondrocytes. The difference of proliferation might be dependent on the difference of Fas-L expression between OA and normal chondrocytes. In conclusion, DcR3 regulated chondrocytes proliferation via ERK signaling and Fas-induced apoptosis. These findings suggest that analysis of DcR3 function could be one of therapeutic strategies for OA treatment. Author contributions Study design: Hayashi, Nishiyama Sample collection: Kuroda, Matsumoto, Fujishiro, Kanzaki, Kurosaka
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Acquisition of data: Hayashi, Hashimoto Analysis and interpretation of the data: Hayashi, Fujishiro, Nishiyama Drafting of the article: Hayashi Critical reading of the manuscript: Nishiyama, Kuroda, Kurosaka Final approval: Nishiyama Conflict of interest All authors do not have any financial support and personal relationships with other people or organization. Acknowledgments We thank Ms Kyoko Tanaka, Ms Minako Nagata, and Ms Masako Sakaguchi for technical assistance, and Ms Janina Tubby for English rewriting. References 1. Pitti RM, Marsters SA, Lawrence DA, Roy M, Kischkel FC, Dowd P, et al. Genomic amplification of a decoy receptor for Fas ligand in lung and colon cancer. Nature 1998;396: 699e703. 2. Bai C, Connolly B, Metzker ML, Hilliard CA, Liu X, Sandig V, et al. Overexpression of M68/DcR3 in human gastrointestinal tract tumors independent of gene amplification and its location in a four-gene cluster. Proc Natl Acad Sci USA 2000;97:1230e5. 3. Yu KY, Kwon B, Ni J, Zhai Y, Ebner R, Kwon BS. A newly identified member of tumor necrosis factor receptor superfamily (TR6) suppresses LIGHT-mediated apoptosis. J Biol Chem 1999;274:13733e6. 4. Migone TS, Zhang J, Luo X, Zhuang L, Chen C, Hu B, et al. TL1A is a TNF-like ligand for DR3 and TR6/DcR3 and functions as a T cell costimulator. Immunity 2002;16:479e92. 5. Hayashi S, Miura Y, Nishiyama T, Mitani M, Tateishi K, Sakai Y, et al. DcR3 expressed in rheumatoid synovial fibroblasts protects the cells against Fas-induced apoptosis. Arthritis Rheum 2007;57:1067e75. 6. Hayashi S, Miura Y, Tateishi K, Takahashi M, Kurosaka M. Decoy receptor 3 is highly expressed in patients with rheumatoid arthritis. Mod Rheumatol 2010;20:63e8. 7. Vignon E, Arlot M, Meunier P, Vignon G. Quantitative histological changes in osteoarthritic hip cartilage: morphometric analysis of 29 osteoarthritic and 26 normal human femoral heads. Clin Orthop 1974;121:269e78. 8. Aigner T, Söder S, Gebhard PM, McAlinden A, Haag J. Mechanisms of disease: role of chondrocytes in the pathogenesis of osteoarthritisestructure, chaos and senescence. Nat Clin Pract Rheumatol 2007;3:391e9. 9. Blanco FJ, Guitian R, Vázquez-Martul E, de Toro FJ, Galdo F. Osteoarthritis chondrocytes die by apoptosis. A possible pathway for osteoarthritis pathology. Arthritis Rheum 1998;41:284e9. 10. Kouri JB, Rosales-Encina JL, Chaudhuri PP, Luna J, Mena R. Apoptosis in human osteoarthritic cartilage: a microscopy report. Med Sci Res 1997;25:245e8. 11. Hsu TL, Chang YC, Chen SJ, Liu YJ, Chiu AW, Chio CC, et al. Modulation of dendritic cell differentiation and maturation by decoy receptor 3. J Immunol 2002;168:4846e53. 12. Santiago B, Galindo M, Palao G, Pablos JL. Intracellular regulation of Fas-induced apoptosis in human fibroblasts by extracellular factors and cycloheximide. J Immunol 2004;172:560e6. 13. Itoh K, Hase H, Kojima H, Saotome K, Nhishioka K, Kobata T. Central role of mitochondria and p53 in Fas-mediated apoptosis of rheumatoid synovial fibroblasts. Rheumatology (Oxford) 2004;43:277e85.
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14. Hashimoto S, Nishiyama T, Hayashi S, Fujishiro T, Takebe K, Kanzaki N, et al. Role of p53 in human chondrocyte apoptosis in response to shear strain. Arthritis Rheum 2009;60:2340e9. 15. Schuerwegh AJ, Dombrecht EJ, Stevens WJ, Van Offel JF, Bridts CH, De Clerck LS. Influence of pro-inflammatory (IL-1 alpha, IL-6, TNF-alpha, IFN-gamma) and anti-inflammatory (IL-4) cytokines on chondrocyte function. Osteoarthritis Cartilage 2003;11:681e7. 16. Hashimoto H, Tanaka M, Suda T, Tomita T, Hayashida K, Takeuchi E, et al. Soluble Fas ligand in the joints of patients with rheumatoid arthritis and osteoarthritis. Arthritis Rheum 1998;41:657e62. 17. Todd AR, Robertson CM, Harwood FL, Sasho T, Williams SK, Pomerleau AC. Characterization of mature vs aged rabbit articular cartilage: analysis of cell density, apoptosis-related gene expression and mechanisms controlling chondrocyte apoptosis. Osteoarthritis Cartilage 2004;12:917e23. 18. Yoon YM, Oh CD, Kim DY, Lee YS, Park JW, Huh TL, et al. Epidermal growth factor negatively regulates chondrogenesis of mesenchymal cells by modulating the protein kinase C-alpha, Erk-1, and p38 MAPK signaling pathways. J Biol Chem 2000;275:12353e9. 19. Pang L, Sawada T, Decker SJ, Saltiel AR. Inhibition of MAP kinase kinase blocks the differentiation of PC-12 cells induced by nerve growth factor. J Biol Chem 1995;270:13585e8.
20. Tateishi K, Miura Y, Hayashi S, Takahashi M, Kurosaka M. DcR3 p.otects THP-1 macrophages from apoptosis by increasing integrin alpha4. Biochem Biophys Res Commun 2009;389:593e8. 21. Yang CR, Wang JH, Hsieh SL, Wang SM, Hsu TL, Lin WW. Decoy receptor 3 (DcR3) induces osteoclast formation from monocyte/macrophage lineage precursor cells. Cell Death Differ 2004;11:S97eS107. 22. Hsu MJ, Lin WW, Tsao WC, Chang YC, Hsu TL, Chiu AW, et al. Enhanced adhesion of monocytes via reverse signaling triggered by decoy receptor 3. Exp Cell Res 2004;292:241e51. 23. You RI, Chang YC, Chen PM, Wang WS, Hsu TL, Yang CY, et al. Apoptosis of dendritic cells induced by decoy receptor 3 (DcR3). Blood 2008;111:1480e8. 24. Tang CH, Hsu TL, Lin WW, Lai MZ, Yang RS, Hsieh SL, et al. Attenuation of bone mass and increase of osteoclast formation in decoy receptor 3 transgenic mice. J Biol Chem 2007;282: 2346e54. 25. Connolly K, Cho YH, Duan R, Fikes J, Gregorio T, LaFleur DW, et al. In vivo inhibition of Fas ligand-mediated killing by TR6, a Fas ligand decoy receptor. J Pharmacol Exp Ther 2001;298: 25e33. 26. Zhang J, Salcedo TW, Wan X, Ullrich S, Hu B, Gregorio T, et al. Modulation of T-cell responses to alloantigens by TR6/DcR3. J Clin Invest 2001;107:1459e68.
DcR3 induces cell proliferation through MAPK signaling in chondrocytes of osteoarthritis S. Hayashi, T. Nishiyama*, Y. Miura, T. Fujishiro, N. Kanzaki, S. Hashimoto, T. Matsumoto, M. Kurosaka, R. Kuroda Department of Orthopaedic Surgery, Kobe University, Graduate School of Medicine, Kobe, Japan
a r t i c l e i n f o
s u m m a r y
Article history: Received 21 January 2011 Accepted 11 March 2011
Introduction: Decoy receptor 3 (DcR3), a soluble receptor belonging to the tumor necrosis factor (TNF) receptor superfamily, competitively binds and inhibits the TNF family including Fas-ligand (Fas-L), lymphotoxin-like inducible protein that competes with glycoprotein D for binding herpesvirus entry mediator on T-cells (LIGHT) and TNF-like ligand 1A (TL1A). In this study, we investigated the functions of DcR3 on osteoarthritis (OA) chondrocytes. Methods: Expressions of DcR3 in chondrocytes were measured by realtime Reverse Transcriptase-Polymerase Chain Reaction (RT-PCR). Expression of DcR3 in sera and joint fluids was measured by enzymelinked immunosorbent assay (ELISA). Chondrocytes were incubated with DcR3-Fc chimera protein (DcR3-Fc) before induction of apoptosis by Fas-L and apoptosis was detected with terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick end labelling labeling (TUNEL) staining and Western blotting of caspase 8 and poly (ADP-ribose) polymerase (PARP). Chondrocytes were incubated with DcR3-Fc and the proliferation was analyzed by 4-[3-(4-iodophenyl)-2-(4-nitrophenyl)-2H-5-tetrazolio]1,3-benzene disulfonate (WST) assay. Phosphorylation of Extracellular Signal-Regulated Kinase (ERK), P38 mitogen-activated protein kinase (MAPK) and Jun N-terminal Kinase (JNK) in chondrocytes was measured by Western blotting after incubation with DcR3-Fc, Mitogen-activated protein kinase kinase (MEK1/2) inhibitor, or P38 MAPK inhibitor. Chondrocytes were treated with DcR3Fc after pre-incubation with blocking antibody of Fas-L, LIGHT and TL1A, and proliferation or phosphorylation of ERK was analyzed. Results: DcR3 was expressed in OA and normal chondrocytes. DcR3-Fc protects chondrocytes from Fasinduced apoptosis. DcR3-Fc increased chondrocytes proliferation and induced the phosphorylation of ERK specifically. DcR3-induced chondrocytes proliferation was inhibited by pre-incubation of PD098059 or blocking Fas-L antibody. DcR3 increased chondrocytes proliferation in OA chondrocytes, but did not in normal. Conclusion: DcR3 regulates the proliferation of OA chondrocytes via ERK signaling and Fas-induced apoptosis. Ó 2011 Osteoarthritis Research Society International. Published by Elsevier Ltd. All rights reserved.
Keywords: Decoy receptor 3 Chondrocyte Apoptosis ERK Fas-ligand Proliferation
Introduction Decoy receptor 3 (DcR3)/TR6/M68 is a member of the tumor necrosis factor receptor (TNFR) superfamily identified1. DcR3 lacks the transmembrane domain of conventional TNFRs and is believed to be a secreted protein1. DcR3 is expressed in some normal tissues
* Address correspondence and reprint requests to: T. Nishiyama, Department of Orthopaedic Surgery, Kobe University, Graduate School of Medicine, 7-5-1 Kusunoki-cho, Chuo-ku, Kobe 650-0017, Japan. Tel: 81-78-382-5985; Fax: 81-78-351-6944. E-mail address: [email protected] (T. Nishiyama).
including colon, stomach, spleen, lymph node, spinal cord, pancreas, and lung1,2. DcR3 is overexpressed in tumor cells including lung and colon cancers1, gliomas, and gastrointestinal tract tumors2. Overexpression of DcR3 might benefit tumors by helping them to avoid the cytotoxic and regulatory effects of Fasligand (Fas-L)1, lymphotoxin-like inducible protein that competes with glycoprotein D for binding herpesvirus entry mediator on T-cells (LIGHT)3, and TNF-like ligand 1A (TL1A)4. Furthermore, DcR3 is expressed in synovial cells of both osteoarthritis (OA) and rheumatoid arthritis (RA)5. Overexpressed DcR3 in rheumatoid synovial cells stimulated with proinflammatory tumor necrosis factor (TNF) alpha protects the cells from apoptosis5,6.
1063-4584/$ e see front matter Ó 2011 Osteoarthritis Research Society International. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.joca.2011.03.005
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OA, the most common degenerative disease of human articular cartilage, is characterized by extracellular matrix damage and an important loss in tissue cellularity7. Cartilage cells are mainly responsible for the anabolicecatabolic balance required for matrix maintenance and tissue function8. OA cartilage has a higher proportion of apoptotic chondrocytes than normal cartilage9 and apoptosis may play a role in diseases involving articular cartilage degeneration10. Therefore, DcR3 might be involved in the regulation of chondrocyte apoptosis. Further, recently, DcR3 was reported to function as a ligand. The expression of Human Leukocyte Antigen DR-1 (HLA-DR) and other costimulatory molecules such as CD40, CD54/ICAM-1, CD1a and CD80/B7.1 was down-regulated by DcR3-Fc and the costimulatory molecule CD86/B7.2 was up-regulated in DCs11. Hence, we speculated that DcR3 might be expressed in chondrocytes and regulate apoptosis and proliferation of chondrocytes possibly resulting in cartilage regeneration. In this study, therefore, we investigated DcR3 expression in chondrocytes and analyzed the function of DcR3 in apoptosis and proliferation, and suggested that DcR3 plays a substantial role in the physiology of OA chondrocytes. Materials and methods Preparation of cartilage Cartilage tissues were obtained during total hip joint replacement surgery from 19 patients with OA. Diagnosis of OA was based on clinical, laboratory, and radiographic evaluations. Normal cartilage tissues were obtained during surgery of femoral neck fracture from seven patients with no history of joint disease and with macroscopically normal cartilage. All the samples were obtained in accordance with the World Medical Association Declaration of Helsinki Ethical Principles for Medical Research Involving Human Subjects. Cell culture Chondrocytes were isolated and cultured from cartilage tissues. Tissues were minced and incubated with trypsin (0.5 mg/ml) (Sigma, St. Louis, MO, USA) for 15 min at 37 C, after which the cartilage was treated with Dulbecco’s modified Eagle’s medium (DMEM; Gibco BR, Grand Island, NY, USA) containing 0.2% of collagenase (Sigma) at 37 C for 15 h. Dissociated cells were cultured in DMEM supplemented with 10% fetal bovine serum (FBS; Biowhittaker, Walkersville, ML, USA) and 100 U/ml of penicillinestreptomycin. After overnight culture, non-adherent cells were removed, and adherent cells were further incubated in fresh medium. All experiments were conducted using first passage cells. Quantification of DcR3, Fas-L and Fas mRNA expression Chondrocytes were cultured in 6-well plates with various stimulations, and RNA was extracted with QIAshredder and RNeasy Mini kit (Qiagen, Hilden, Germany) according to the manufacturer’s protocol. 1 mg of total RNA was reverse-transcribed to first-strand cDNA with 1.25 mM oligo dT primer in 40 ml PCR buffer II containing 2.5 mM MgC12, 0.5 mM dNTP mix, 0.5 U RNase inhibitor, and 1.25 U MuLV reverse transcriptase (Perkin Elmer, Foster City, CA, USA) at 42 C for 60 min. The relative expression levels of mRNA encoding DcR3, Fas-L or Fas in chondrocytes were analyzed by TaqManÒ realtime PCR using an ABI prism 7500 sequence detection system (Applied Biosystems, Foster City, CA, USA). Pre-designed primers and probes for human DcR3, Fas-L, Fas and human glyceraldehyde-3-phosphate
dehydrogenase (GAPDH) to be used as the control were obtained from Applied Biosystems. Quantification of DcR3 protein concentration in sera or joint fluid In order to measure DcR3 in serum and joint fluid, we used the human DcR3 enzyme-linked immunosorbent assay (ELISA) kit (Bender Med Systems, Vienna, Austria) according to the manufacturer’s protocol. Briefly, 100 ml of each sample were incubated in duplicate in microplates coated with anti-DcR3 monoclonal antibody for 2 h. Following incubation, biotin-conjugate anti-DcR3 monoclonal antibody was added and incubated as a primary antibody for 2 h. After the microplate had been washed, streptavidinhorseradish peroxidase (HRP) was added and incubated for 1 h. After washing off any unbound streptavidin-HRP, tetramethylbenzidine was added as a substrate and the absorbance was measured at 450 nm after 20 min in a microplate reader (BioTek Instruments, Burlington, VT, USA). The absorbance of each sample was plotted against a standard curve produced by serial dilutions of recombinant human DcR3-Fc in duplicate. The concentrations of DcR3 in serum and joint fluid were calculated by logarithmic analysis. All samples with an absorbance of less than zero according to the standard curve were discarded from the final analysis. Fas-induced apoptosis in chondrocytes Before the induction of apoptosis, chondrocytes were pretreated with 0.1, 1, 10 and 100 ng/ml of recombinant human DcR3Fc chimera protein (DcR3-Fc) (R&D Systems) for 24 h in Opti Minimum essential medium (MEM) (Invitrogen, Carlsbad, CA, USA). DcR3-Fc and recombinant DcR3 protein (rDcR3) have the same binding affinity to LIGHT and the same inhibitory effect on the development of Cytotoxic T-lymphocyte (CTL) in mice indicating that the biological effect of DcR3-Fc is equivalent to rDcR312. Fas-induced apoptosis was introduced by incubation with 100 ng/ml of recombinant human Fas-ligand (hFas-L) (R&D Systems) for an additional 12 h. Fas-induced apoptotic signals were confirmed by terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick end labeling (TUNEL) staining and the detection of cleavage of caspase 813 and poly (ADP-ribose) polymerase (PARP)9. Proliferation assay Chondrocytes were seeded in 96-well plates at a density of 5 103 cells per well in DMEM medium supplemented with 10% FBS. After plating cells for overnight, chondrocytes were treated with DcR3-Fc or IgG-Fc (Millipore, Billerica, MA, USA) as control to eliminate the effects of Fc. In some cases, chondrocytes were preincubated with blocking antibody of Fas-L (BD Pharmingen, San Jose, CA, USA), LIGHT and TL1A (Biolegend, San Diego, CA, USA) or IgG (Acris, Littleton, CO, USA) as control for 12 h. Cell proliferation was assessed using Cell-Counting Kit-8 (Dojindo, Kumamoto, Japan) (4-[3-(4-iodophenyl)-2-(4-nitrophenyl)-2H-5-tetrazolio]1,3-benzene disulfonate (WST) assay) according to the manufacturer’s instruction. The principle of WST assay is following. Tetrazolium salt-8 (WST-8) is cleaved to soluble formazan dye by the succinate-tetrazolium reductase, which exists in mitochondrial respiratory chain and is active only in viable cells. Total activity of this mitochondrial succinate-tetrazolium reductase in a sample rises with the increase of viable cells. The absorbance of formazan dye solution is in direct proportion to the number of viable cells. Therefore, this assay is designed for non-radioactive quantification of cell growth and viability in proliferation. The values, corresponding to the number of viable cells, were read at OD 450 nm with Microplate Reader (BioTek Instrument, Winooski, VT, USA).
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DcR3-Fc and rDcR3 have the same binding affinity to LIGHT and the same inhibitory effect on the development of CTL in mice indicating that the biological effect of DcR3-Fc is equivalent to rDcR312. Western blotting Chondrocytes were washed three times with Phosphate Buffered Saline (PBS) and lysed in hypotonic lysis buffer (25 mM Tris, 1% NP-40, 150 mM NaCl, 1.5 mM Ethylene glycol tetraacetic acid (EGTA)) supplemented with protease and phosphatase inhibitor mix (Roche Diagnostics, Basel, Switzerland) on ice for 20 min. The lysates were centrifuged at 15,000 rpm for 20 min to remove cellular debris and the supernatants were collected. Cytoplasmic proteins were quantified by the Bradford method with protein assay reagent (BioRad, Hercules, CA, USA), and diluted to an equal concentration with hypotonic buffer. Each sample was mixed with 3 electrophoresis sample buffer (BioRad) and electrophoresed on 7.5e15% polyacrylamide gradient gel (Biocraft, Tokyo, Japan) and transblotted electrically onto the blotting membrane (GE Healthcare, Connecticut, CT, USA). The expression of DcR3 protein was detected using rabbit antihuman DcR3 polyclonal Ab (R&D systems). The expression of caspase 8 was detected using mouse anti-human caspase 8 monoclonal Ab (Cell Signaling Technology, Beverly, MA, USA). The expression of PARP was detected using rabbit anti-human PARP polyclonal Ab (Cell Signaling Technology). The expression of phospho-Extracellular Signal-Regulated Kinase (ERK) or ERK was detected using mouse anti-human phospho-ERK Ab (Cell Signaling Technology) or rabbit anti-human ERK Ab (Cell Signaling Technology). The
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expression of phospho-P38 mitogen-activated protein kinase (MAPK) or P38 MAPK was detected using rabbit anti-human phospho-P38 MAPK Ab (Cell Signaling Technology) or rabbit anti-human P38 MAPK Ab (Cell Signaling Technology). The expression of phospho-Jun N-terminal Kinase (JNK) or JNK was detected using rabbit anti-human phospho-JNK Ab (Cell Signaling Technology) or rabbit anti-human JNK Ab (Cell Signaling Technology). HRP conjugated goat anti-rabbit IgG Ab (GE healthcare) or HRP conjugated rabbit anti-mouse IgG Ab (GE healthcare) was used for a secondary antibody, and the signals were visualized by ECL plus reagent (GE healthcare) with Chemilumino analyzer LAS-3000 mini (FUJI FILM, Tokyo, Japan). The expression of proteins was determined by semi-quantification of digitally captured image using the public domain NIH Image program (developed at the U.S. National Institutes of Health and available on the Internet at http://rsb.info.nih.gov/nihimage/). Values were normalized to alpha-tublin expression. TUNEL staining 2 105 chondrocytes were cultured in 8-well chamber slides (Nunc Roskilde, Denmark). After various stimulations, the chondrocytes were fixed with 4% neutral buffered formalin for 10 min and apoptotic cells were determined using TUNEL assay kit (Wako) according to the manufacturer’s protocol. Statistical analysis Statistical analysis was performed using one-way analysis of variance, with Tukey’s post hoc test for multiple comparisons of paired samples. Results are presented as mean values with 95%
Fig. 1. (a) Expression levels of DcR3, Fas-L or Fas mRNA were quantified by realtime PCR. Expression ratios of DcR3, Fas or Fas-L (OA chondrocytes/Normal chondrocytes) are shown. Columns represent mean values with 95% CI. The results shown are the average of seven individual samples. Values were normalized to GAPDH expression. (b) Human Serum or joint fluid levels of DcR3 protein were measured by ELISA. Control sera was collected from 10 healthy subjects, OA sera and joint fluid were from 10 subjects with OA. Columns represent mean values with 95% CI.
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Fig. 2. (a and b) Effect of hFas-L and DcR3-Fc protein for Fas-induced apoptosis of chondrocytes. (a) Columns represent mean values with 95% CI. Columns represent of TUNEL positive cells, at least 300 cells were counted by a blinded observer in the same experiment. The results shown are the average of four individual samples. (b) Expressions of cleaved caspase 8 and PARP were analyzed by Western blotting. The results shown represent three independent experiments.
confidence intervals (95% CI), and were considered statistically significant for P < 0.05. Results
between OA (mean ¼ 0.924, 95% CI ¼ 0.56e1.29, N ¼ 7) and normal chondrocytes (mean ¼ 1, 95% CI ¼ 0.78e1.22, N ¼ 7). However, the levels of Fas-L were significantly higher in OA (mean ¼ 2.97, 95% CI ¼ 2.14e3.79, N ¼ 7) than in normal chondrocytes (mean ¼ 1, 95% CI ¼ 0.76e1.24, N ¼ 7) (Fig. 1a).
Expression levels of DcR3 mRNA were not changed between OA and normal chondrocytes
DcR3 is expressed in joint fluids of patients with OA
In order to compare the expression levels of DcR3, Fas-L or Fas mRNA between OA and normal chondrocytes, we analyzed mRNA levels by realtime Reverse Transcriptase-Polymerase Chain Reaction (RT-PCR). The expression levels of DcR3 were not significantly changed between OA (mean ¼ 0.89, 95% CI ¼ 0.50e1.28, N ¼ 7) and normal chondrocytes (mean ¼ 1, 95% CI ¼ 0.78e1.22, N ¼ 7). The expression levels of Fas were not significantly changed
In order to analyze the expression of DcR3 protein in sera and joint fluid, we quantified the concentration of DcR3 by DcR3 ELISA kit. The average concentration of serum DcR3 was 0.33 ng/ml in OA patients (95% CI ¼ 0.04e0.62, N ¼ 10), and 0.24 ng/ml in healthy donor (95% CI ¼ 0.03e0.45, N ¼ 10) (Fig. 1b). There was no significant difference between OA and healthy subjects. While, the average concentration of DcR3 in joint fluid of OA patients was
Fig. 3. (a) Effect of DcR3 on chondrocyte proliferation analyzed by WST assay. Chondrocytes were incubated with 5 mg/ml of IgG-Fc or 1, 5 mg/ml of DcR3-Fc for 48 h. Columns show the ratio of OD 450 nm value in comparison with untreated chondrocytes. The results shown are the average of four individual samples. Columns represent mean values with 95% CI. (b) Effect of DcR3-Fc for ERK, P38 MAPK, or JNK phosphorylation analyzed by Western blotting. Chondrocytes were incubated with 5 mg/ml of DcR3-Fc or 5 mg/ml of IgG-Fc for 0, 5, 10 and 30 min. The results shown represent three independent experiments.
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Fig. 4. Effects of PD098059 (PD) and SB203580 (SB) against DcR3-induced proliferation of chondrocytes. (a) Chondrocyte proliferation was analyzed by WST assay. Chondrocytes were incubated with 5 mg/ml of IgG-Fc, 5 mg/ml of DcR3-Fc, 50 mM of PD098059 (R&D) or 10 mM of SB203580 (R&D) for 48 h. In some cases, chondrocytes were pre-treated with 50 mM of PD098059 or 10 mM of SB203580 for 30 min, and incubated with 5 mg/ml of DcR3-Fc for 48 h. Columns show the ratio of OD 450 nm value in comparison with untreated chondrocytes. The results shown are the average of three individual samples. Columns represent mean values with 95% CI. (b) Phosphorylation of ERK was analyzed by Western blotting. Chondrocytes were incubated with 5 mg/ml of IgG-Fc, 5 mg/ml of DcR3-Fc, 50 mM of PD098059 or 10 mM of SB203580 for 10 min. In some cases, chondrocytes were pretreated with 50 mM of PD098059 or 10 mM of SB203580 for 30 min, and incubated with 5 mg/ml of DcR3-Fc for 10 min. The results shown represent three independent experiments.
Fig. 5. Effects of blocking antibody of Fas-L, LIGHT and TL1A for DcR3-induced chondrocytes proliferation. (a) Chondrocyte proliferation was analyzed by WST assay. Chondrocytes were pre-incubated with 100 ng/ml of blocking antibody of Fas-L, 5 mg/ml of blocking antibody of LIGHT and TL1A or 5 mg/ml of IgG as control for 12 h. After washing cells with PBS, chondrocytes were incubated with 5 mg/ml of DcR3-Fc for 48 h. Columns show the ratio of OD 450 nm value in comparison with 5 mg/ml of IgG pre-treatment. The results shown are the average of four individual samples. Columns represent mean values with 95% CI. (b) Phosphorylation of ERK was analyzed by Western blotting. After pre-incubation with IgG, blocking antibody of Fas-L, LIGHT and TL1A for 12 h, chondrocytes were washed with PBS and incubated with 5 mg/ml of DcR3-Fc for 0, 5, 10 and 30 min.
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1.44 ng/ml (95% CI ¼ 0.90e2.00, N ¼ 10). The average concentration of DcR3 in joint fluid of OA patients was much higher than in sera (Fig. 1b). DcR3 protein inhibited Fas-induced apoptosis in chondrocytes TUNEL positive apoptotic cells induced by Fas-L were significantly decreased in a dose dependent manner when chondrocytes were pre-incubated with DcR3-Fc protein (Untreated: mean ¼ 8, 95% CI ¼ 5.7e10.3, N ¼ 4) (0 ng/ml DcR3Fc: mean ¼ 44.6, 95% CI ¼ 35.5e53.8, N ¼ 4) (0.1 ng/ml DcR3-Fc: mean ¼ 34.9, 95% CI ¼ 31.8e38.1, N ¼ 4) (1 ng/ml DcR3Fc: mean ¼ 30.2, 95% CI ¼ 28.0e32.4, N ¼ 4) (10 ng/ml DcR3-Fc: mean ¼ 24.3, 95% CI ¼ 17.8e30.7, N ¼ 4) (100 ng/ml DcR3Fc: mean ¼ 13.9, 95% CI ¼ 5.7e22.1, N ¼ 4) (Fig. 2a). The cleavage of caspase 8 and PARP was increased by 100 ng/ml of Fas-L treatment. However, the cleavage of caspase 8 and PARP with Fas-L treatment was inhibited by pre-treatment with 100 ng/ml of DcR3-Fc (Fig. 2b). DcR3 increased OA chondrocytes proliferation via ERK phosphorylation WST assay revealed that chondrocytes proliferation were increased by the treatment of DcR3-Fc in a dose dependent manner (Untreated: mean ¼ 1, 95% CI ¼ 0.94e1.07, N ¼ 4) (IgG-Fc: mean ¼ 1.04, 95% CI ¼ 0.86e1.22, N ¼ 4) (1 mg/ml DcR3Fc: mean ¼ 1.20, 95% CI ¼ 1.11e1.30, N ¼ 4) (5 mg/ml DcR3-Fc: mean ¼ 1.32, 95% CI ¼ 1.24e1.39, N ¼ 4) (Fig. 3a). In order to investigate the signal transduction of DcR3, we tested phosphorylation of ERK, P38 MAPK and JNK in chondrocytes with the treatment of 5 mg/ml of DcR3-Fc. Western blotting revealed that phosphorylation of ERK was increased to 12.63 folds of untreated samples at 10 min and 3.12 folds at 30 min by treatment with 5 mg/ml of DcR3-Fc (Fig. 3b). While, neither P38 MAPK nor JNK signaling was activated by treatment with 5 mg/ml of DcR3-Fc for up to 30 min (Fig. 3b). Suppression of ERK activity inhibited DcR3-induced chondrocytes proliferation WST assay revealed that the effect of DcR3-Fc for chondrocytes proliferation was inhibited by treatment with specific Mitogen-
activated protein kinase kinase (MEK1/2) inhibitor PD098059, but not by specific P38 MAPK inhibitor SB203580 (Untreated: mean ¼ 1, 95% CI ¼ 0.91e1.09, N ¼ 3) (IgG-Fc: mean ¼ 1.01, 95% CI ¼ 0.93e1.10, N ¼ 3) (PD: mean ¼ 0.96, 95% CI ¼ 0.90e1.02, N ¼ 3) (SB: mean ¼ 1.04, 95% CI ¼ 0.99e1.09, N ¼ 3) (DcR3-Fc: mean ¼ 1.36, 95% CI ¼ 1.16e1.60, N ¼ 3) (DcR3-Fc þ PD: mean ¼ 1.10, 95% CI ¼ 0.94e1.26, N ¼ 3) (DcR3-Fc þ SB: mean ¼ 1.31, 95% CI ¼ 1.22e1.41, N ¼ 3) (Fig. 4a). Western blotting confirmed that the activation of ERK signaling by treatment with 5 mg/ml DcR3-Fc was inhibited by PD098059, but not by SB203580 (Fig. 4b).
Anti-Fas-L antibody inhibited the DcR3-induced chondrocytes proliferation through ERK signaling In order to figure out the mechanism of DcR3-induced signal transduction in chondrocytes proliferation, we tested the effect of blocking antibody of Fas-L, LIGHT and TL1A for DcR3-induced proliferation. WST assay revealed that the effect of DcR3-Fc for chondrocytes proliferation was inhibited by pre-incubation with blocking antibody of Fas-L, but not by blocking antibody of LIGHT or TL1A (IgG: mean ¼ 1, 95% CI ¼ 0.98e1.02, N ¼ 4) (Fas-L: mean ¼ 0.61, 95% CI ¼ 0.56e0.67, N ¼ 4) (LIGHT: mean ¼ 1.03, 95% CI ¼ 0.71e1.36, N ¼ 4) (TL1A: mean ¼ 0.99, 95% CI ¼ 0.98e1.02, N ¼ 4) (Fig. 5a). Western blotting showed that phosphorylation of ERK by treatment with 5 mg/ml of DcR3-Fc was inhibited by preincubation with blocking antibody of Fas-L, but not with blocking antibody of LIGHT or TL1A (Fig. 5b).
DcR3 did not increase proliferation of normal chondrocytes In order to compare the difference between OA and normal chondrocytes in response to DcR3 treatment, we tested chondrocytes proliferation by treatment of 5 mg/ml of DcR3-Fc in OA and normal chondrocytes. WST assay revealed that DcR3-Fc increased chondrocytes proliferation in OA chondrocytes, but did not in normal (Untreated normal: mean ¼ 1, 95% CI ¼ 0.87e1.13, N ¼ 4) (Untreated OA: mean ¼ 1, 95% CI ¼ 0.82e1.18, N ¼ 4) (IgG-Fc normal: mean ¼ 0.95, 95% CI ¼ 0.88e1.02, N ¼ 4) (IgG-Fc OA: mean ¼ 1.03, 95% CI ¼ 0.94e1.12, N ¼ 4) (DcR3-Fc normal: mean ¼ 1.09, 95% CI ¼ 0.88e1.30, N ¼ 4) (DcR3-Fc OA: mean ¼ 1.31, 95% CI ¼ 1.27e1.34, N ¼ 4) (Fig. 6).
Fig. 6. Comparison of DcR3 effect on proliferation between OA and normal chondrocytes. Chondrocytes were incubated with 5 mg/ml of IgG-Fc or DcR3-Fc for 48 h. Chondrocytes proliferation was analyzed by WST assay. Columns show the ratio of OD 450 nm value in comparison with untreated chondrocytes. White panels: Normal chondrocytes. Black panels: OA chondrocytes. The results shown are the average of four individual samples. Columns represent mean values with 95% CI.
S. Hayashi et al. / Osteoarthritis and Cartilage 19 (2011) 903e910
Discussion A significant body of knowledge about the mechanism of chondrocytes apoptosis was reported. Many manuscripts reported that chondrocytes apoptosis were induced via mitochondrial pathway or cytokines like TNF alpha14,15. We also have shown that mechanical stress induced chondrocytes apoptosis via p53-mitochondrial pathway14. While, it has been reported that several stimuli including Fas-L16 induced apoptosis in chondrocytes and a significant amount of FasL was found in synovial fluids from patients with OA16. Expression of Fas-L was higher in aged cartilage than in mature cartilage, which correlated with the decrease in viable cell density in rabbit articular cartilage during aging17. We confirmed that the expression of Fas-L was increased in OA chondrocytes. These findings suggest that Fas/ Fas-L is an important molecule for the regulation of apoptosis of chondrocytes in OA cartilage. DcR3 is expressed in a variety of tissues and tumors1,2, and regulates cell proliferation, however, the expression of DcR3 in chondrocytes was not investigated previously. In this study, we demonstrated that DcR3 is expressed in normal and OA chondrocytes similarly, and the expression levels of DcR3 protein in the joint fluid were higher than in sera. Average concentration of DcR3 in joint fluid of OA patients was 1.44 ng/ml. While, we also demonstrated that 1 ng/ml of DcR3-Fc inhibited Fas-induced apoptosis in chondrocytes. These results suggest that DcR3 protein is secreted into the joint and actually protects chondrocytes from death via Fas-induced apoptosis in physiological circumstance. However, the amount of DcR3 may be insufficient to bind to the increased amount of Fas-L in OA cartilage16,17 and incapable to inhibit Fas-induced apoptosis. From the standpoint of chondrocyte proliferation, MAPK signaling is one of the essential mitogenic signaling in many kinds of cells and plays a key role in a variety of cellular responses including proliferation, differentiation and apoptosis of cells18,19. We recently demonstrated that DcR3 has functions as a ligand to inhibit apoptosis in THP-1 macrophages by increasing very late antigen 4 (VLA4) expression20. Several studies have reported that soluble DcR3 activates reverse signaling as a ligand, inducing osteoclast formation from monocytes/macrophages via ERK and p38 MAPK signaling21, inducing actin reorganization and enhancing adhesion of monocytes22, and inducing dendritic cell apoptosis by activating Protein Kinase C (PKC-d) and JNK23,24. In this study we demonstrated that DcR3-Fc increased proliferation of chondrocytes through activation of ERK signaling pathway specifically, but not in p38 MAPK or JNK. DcR3-Fc fusion protein has been shown to have similar binding affinity and specificity as DcR325,26, and in our experiments control IgG-Fc had no effect on chondrocytes. Our results therefore are the first to suggest that DcR3 may regulate chondrocytes proliferation by binding Fas-L through ERK signaling. We further demonstrated that chondrocytes proliferation by DcR3-Fc treatment was different between OA and normal chondrocytes. The difference of proliferation might be dependent on the difference of Fas-L expression between OA and normal chondrocytes. In conclusion, DcR3 regulated chondrocytes proliferation via ERK signaling and Fas-induced apoptosis. These findings suggest that analysis of DcR3 function could be one of therapeutic strategies for OA treatment. Author contributions Study design: Hayashi, Nishiyama Sample collection: Kuroda, Matsumoto, Fujishiro, Kanzaki, Kurosaka
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Acquisition of data: Hayashi, Hashimoto Analysis and interpretation of the data: Hayashi, Fujishiro, Nishiyama Drafting of the article: Hayashi Critical reading of the manuscript: Nishiyama, Kuroda, Kurosaka Final approval: Nishiyama Conflict of interest All authors do not have any financial support and personal relationships with other people or organization. Acknowledgments We thank Ms Kyoko Tanaka, Ms Minako Nagata, and Ms Masako Sakaguchi for technical assistance, and Ms Janina Tubby for English rewriting. References 1. Pitti RM, Marsters SA, Lawrence DA, Roy M, Kischkel FC, Dowd P, et al. Genomic amplification of a decoy receptor for Fas ligand in lung and colon cancer. Nature 1998;396: 699e703. 2. Bai C, Connolly B, Metzker ML, Hilliard CA, Liu X, Sandig V, et al. Overexpression of M68/DcR3 in human gastrointestinal tract tumors independent of gene amplification and its location in a four-gene cluster. Proc Natl Acad Sci USA 2000;97:1230e5. 3. Yu KY, Kwon B, Ni J, Zhai Y, Ebner R, Kwon BS. A newly identified member of tumor necrosis factor receptor superfamily (TR6) suppresses LIGHT-mediated apoptosis. J Biol Chem 1999;274:13733e6. 4. Migone TS, Zhang J, Luo X, Zhuang L, Chen C, Hu B, et al. TL1A is a TNF-like ligand for DR3 and TR6/DcR3 and functions as a T cell costimulator. Immunity 2002;16:479e92. 5. Hayashi S, Miura Y, Nishiyama T, Mitani M, Tateishi K, Sakai Y, et al. DcR3 expressed in rheumatoid synovial fibroblasts protects the cells against Fas-induced apoptosis. Arthritis Rheum 2007;57:1067e75. 6. Hayashi S, Miura Y, Tateishi K, Takahashi M, Kurosaka M. Decoy receptor 3 is highly expressed in patients with rheumatoid arthritis. Mod Rheumatol 2010;20:63e8. 7. Vignon E, Arlot M, Meunier P, Vignon G. Quantitative histological changes in osteoarthritic hip cartilage: morphometric analysis of 29 osteoarthritic and 26 normal human femoral heads. Clin Orthop 1974;121:269e78. 8. Aigner T, Söder S, Gebhard PM, McAlinden A, Haag J. Mechanisms of disease: role of chondrocytes in the pathogenesis of osteoarthritisestructure, chaos and senescence. Nat Clin Pract Rheumatol 2007;3:391e9. 9. Blanco FJ, Guitian R, Vázquez-Martul E, de Toro FJ, Galdo F. Osteoarthritis chondrocytes die by apoptosis. A possible pathway for osteoarthritis pathology. Arthritis Rheum 1998;41:284e9. 10. Kouri JB, Rosales-Encina JL, Chaudhuri PP, Luna J, Mena R. Apoptosis in human osteoarthritic cartilage: a microscopy report. Med Sci Res 1997;25:245e8. 11. Hsu TL, Chang YC, Chen SJ, Liu YJ, Chiu AW, Chio CC, et al. Modulation of dendritic cell differentiation and maturation by decoy receptor 3. J Immunol 2002;168:4846e53. 12. Santiago B, Galindo M, Palao G, Pablos JL. Intracellular regulation of Fas-induced apoptosis in human fibroblasts by extracellular factors and cycloheximide. J Immunol 2004;172:560e6. 13. Itoh K, Hase H, Kojima H, Saotome K, Nhishioka K, Kobata T. Central role of mitochondria and p53 in Fas-mediated apoptosis of rheumatoid synovial fibroblasts. Rheumatology (Oxford) 2004;43:277e85.
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