Nitrate/nitrite colorimetric assay kits were purchased from Cayman Chemical (Ann Arbor, MI, USA). Ly83583 was purchased from Calbiochem (San Diego, CA, USA), 1-hydroxy-2-oxo-3-(3-aminopropyl)-3-isopropyl-1-triazene (NOC-5), SNAP, SB202190, PD98059, MG132 and Bay 11-7082 were from Alexis (Carlsbad, CA, USA), and zinc protoporphyrin (ZnPP) and cobalt protoporphyrin (CoPP) were from Frontier Scientific (Logan, UT, USA). Anti-phospho extracellular signal-regulated protein kinase (ERK) 1/2, anti-phospho-p38, anti-ERK 1/2, and anti-p38 were purchased from New England Biolabs (Beverly, MA, USA), anti-Bcl-2 from Transduction Laboratories (Lexington KY, USA), anti-Bax from Pharmingen (San Diego, CA, USA), and anti-heme oxygenase 1 (anti-HO-1), anti-Bcl-XL, anti-Mcl-1, anti-p53, anti-CIAP1 and anti-CIAP2 from Santa Cruz (Santa Cruz, CA, USA). All other reagents were obtained from Sigma (St Louis, MO, USA) unless specified otherwise.
Cartilage samples were obtained from the femoral condyle and from the tibial plateau of the knees of osteoarthritis patients at the time of joint replacement surgery. All cartilage samples were procured after obtaining oral informed consent from the patients and institutional approval. Pieces of articular cartilage were cut, minced, and incubated sequentially with pronase and collagenase in DMEM until they had been digested. Released cells were seeded at 4 × 106/plate in 10 cm culture plates in DMEM supplemented with 10% FCS, 1% L-glutamine, and 1% Fungizone (Gibco, Grand Island, NY, USA) and in DMEM supplemented with penicillin/streptomycin (150 units/ml and 50 mg/ml, respectively). Confluent chondrocytes were split once after about 7 days and were seeded at high density, and these first-passage adherent chondrocytes were then used in subsequent experiments.
NOC-5 was dissolved in 10 mM NaOH to produce a 200 mM stock solution and was stored at -20°C. SNP, 3-morpholinosydnonimine (SIN-1), and SNAP were freshly dissolved in water before each experiment. All NO donor compounds were diluted with DMEM and added directly to cultured chondrocytes. The final products of NO in vivo are nitrite and nitrate, the sum of which can be used as an index of total NO production. Chondrocyte culture media were harvested after being incubated for 24 hours with the respective NO donors, and were then analyzed using a nitrate/nitrite colorimetric assay kit as recommended by the manufacturer. Briefly, nitrate was converted to nitrite using nitrate reductase, and then Griess reagents were added to form a deep-purple azo compound. Absorbance was measured at 540 nm using a plate reader to determine the nitrite concentrations. The detection limit of the assay was 1 μM.
Quantification and verification of cell death
Cell death was quantitated using the 3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyltetrazdium bromide (MTT) assay, as previously described . Briefly, chondrocytes were seeded at 4 × 104/100 μl/well in 96-well microtiter plates. Cell death was induced by treating with 1 mM SNP. To protect cells, chondrocytes were treated with 0.1 mM SNP, 50 μM CoPP, or 1 mM dibutylyl guanosine-3',5'-cyclic monophosphate (DBcGMP) 14 hours prior to being treated with 1 mM SNP. MTT was then added to each well to a final concentration of 0.125 mg/ml after they had been incubated with 1 mM SNP for 24 hours, and plates were incubated at 37°C for a further 3 hours. The formazan product obtained was solubilized with 100 μl dimethylsulfoxide and optical densities were read at 595 nm. Percentage cell survival was calculated by taking the optical density of cells post-treatment, dividing this by the optical density of the untreated control cells, and multiplying by 100. Cell death was also verified by flow cytometry. Chondrocytes were trypsinized after treatment and were sedimented, and the cell pellets obtained were washed and stained with 100 μg/ml propidium iodide solution for 15 min. For each sample, 104 cells were analyzed by FACS II flow cytometry (Becton Dickinson, Mountain View, CA, USA).
Cellular proteins were extracted in lysis buffer containing 50 mM sodium acetate, pH 5.8, 10% v/v SDS, 1 mM ethylene diaminetetraacetic acid, 1 mM phenylmethylsulfonyl fluoride, and 1 μg/ml aprotinin at 4°C. Samples were electrophoresed on 12% SDS-polyacrylamide gel, and transferred to polyvinylidene difluoride membranes. Blots were blocked with Tris-buffered saline containing 5% non-fat milk at room temperature for 1 hour, and then incubated with the respective antibodies overnight at 4°C. Finally, blots were incubated with 1:5000 peroxidase-conjugated goat anti-mouse or anti-rabbit IgG (Biorad, Hercules, CA, USA) for 1 hour. Bound immunoglobulin was detected by enhanced chemiluminescence (Amersham, Bucks, UK).
Electrophoretic mobility shift assay
Nuclear extracts from chondrocytes were prepared from 2 × 106 cells, as described previously with minor modification . Briefly, cells were incubated on ice for 15 min with homogenization buffer containing 10 mM HEPES-KOH, 4 mM MgCl2, 10 mM KCl, 1 mM NaF, 0.5 mM dithiothreitol, 1 mM phenylmethylsulfonyl fluoride, and 20 μg/ml leupeptin. After adding detergent, the lysates were centrifuged at 3000g for 5 min. Pellets were resuspended in extraction buffer containing 20 mM HEPES-KOH, 1.5 mM MgCl2, 420 mM NaCl, 25% glycerol, 1 mM NaF, 0.5 mM dithiothreitol, 1 mM phenylmethylsulfonyl fluoride, 20 μg/ml leupeptin, and 0.2 mM ethylene diaminetetraacetic acid. After incubation on ice and centrifugation, supernatants were collected, the protein content was measured, and 5 μg portions of extracts were used for the binding reaction. A consensus double-stranded NF-κB probe was obtained from Promega (Madison, WI, USA), and was end-labeled using γ-32P-adenosine-5-triphosphate. After incubating nuclear extracts in 2 μl gel binding buffer (Promega), end-labeled probe was added (100,000 cpm/sample). Samples were then incubated for 20 min and were loaded onto 4% nondenaturing polyacrylamide gels. Electrophoresis was run for 3 hours at 4°C. Protein complexes were identified by autoradiography.
Data are expressed as means ± standard deviations. The paired t test was used to compare controls and treatment conditions, and significance was accepted at a confidence level of 95% (P < 0.05).