Culture media and fetal calf serum (FCS) were from Gibco BRL (Paisley, UK). Culture flasks were purchased from Costar (Cambridge, MA, USA). Unless indicated, the rest of chemicals and enzymes were obtained from Sigma-Aldrich (St. Louis, MO). Antibodies against human Hsp90β (sc-1057), α-tubulin (sc-5286), the peroxidase-conjugated secondary antibodies and the FITC-conjugated anti-goat secondary antibody were from Santa Cruz Biotechnology (Sta. Cruz, CA, USA).
Cartilage procurement and processing
Macroscopically normal human knee cartilage from adult donors from both genders (mean age 60.3 years; age range 54-65 years) without history of joint disease was provided by the Tissue Bank and the Autopsy Service at Hospital Universitario A Coruña. Osteoarthritic cartilage was obtained from patients diagnosed with OA according to the American College of Rheumatology (ACR) criteria, which underwent joint surgery (mean age 64.6 years; age range 52-71 years). Knee radiographs from the OA participants were classified as grade IV according to the Kellgren and Lawrence (K/L) scoring system. All patients have signed the informed consent and the project was approved by the Regional Ethical Committee from Galicia (Spain). Once cartilage surfaces were rinsed with saline, scalpels were used to cut parallel sections 5 mm apart, vertically from the cartilage surface onto the subchondral bone. These cartilage strips were then cut-off from this bone, and the tissue was incubated with trypsin at 37°C for 10 minutes. After removing trypsin solution, the cartilage slices were treated for 12-16 h with type IV clostridial collagenase in Dulbecco's modified Eagle's medium (DMEM) with 5% FCS in order to release cartilage cells.
Primary culture of chondrocytes
Chondrocytes were recovered and plated at high density (4 × 106 per 162-cm2 flask) in DMEM supplemented with 100 units/mL penicillin, 100 μg/mL streptomycin, 1% glutamine and 10% FCS. The cells were incubated at 37°C in a humidified gas mixture containing 5% CO2 balanced with air. Chondrocytes were used at weeks 2-3 at confluency in primary culture. Cell viability was assessed by trypan blue dye exclusion.
Protein sample preparation
Chondrocytes (3-5 × 106 cells) were recovered from culture flasks by trypsinization and washed twice in a saline buffer containing 130 mM NaCl, 5 mM KCl, 2.5 mM Tris HCl (pH 7.5) and 0.7 mM Na2HPO4. Cells were then transferred to microfuge tubes, where cell pellets were solubilized by vortexing and one hour incubation with gentle agitation in 200 μl of an isolectric focusing-compatible lysis buffer containing 8.4 M urea, 2.4 M thiourea, 5% cholamidopropyl diethylamoniopropane sulfonate (CHAPS), 1% carrier ampholytes (IPG Buffer pH 3-10 NL), 0.4% Triton X-100 and 2 mM dithiothreitol (DTT).
For protein quantification, 10 μl of the protein extract were diluted 10x with water and precipitated for at least 1 h with 0.02% sodium deoxycholate and 10% trichloroacetic acid. The precipitate was washed once with 2 volumes of ice-cold acetone, allowed to dry, and solubilized in alkaline SDS (5% SDS, 0.1 N NaOH). 5 to 10 μl of this sample were employed to quantify total chondrocytic proteins in each lysate by the BCA technique (Pierce Perbio, Rockford, IL, USA).
Cell viability assay
Cell viability was evaluated by Trypan blue dye exclusion throughout the work. Nevertheless, due to the high amount of NO accumulation observed in chondrocytes after Novobiocin treatment, we decided to quantify cell viability with a more accurate method in these experiments. The MTS [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2Htetrazolium, inner salt] assay was employed with this aim, using the CellTiter 96® AQueous Non-Radioactive Cell Proliferation Assay (Promega, Wisconsin, USA) kit and used following the manufacturer's instructions. Chondrocytes were seeded into 96-well plates at a density of 2 × 103 per well (100 μl). This cell number was empirically determined for our cells by performing a cell titration assay. Briefly, human chondrocytes (0.5 × 103 - 1 × 104) in DMEM supplemented with 10% FBS were seeded onto a 96-well plate. The medium was allowed to equilibrate for 24 hours, and then 10 μl of combined MTS/PMS (phenazine methosulfate) solution were added to each well. After 3 h at 37°C in a humidified, 5% CO2 atmosphere, the absorbance at 490nm was recorded using an ELISA plate reader (Labsystems Multiskan Plus Plate Reader). Once the optimal cell number was determined (near the low end of the linear range of the assay), chondrocytes were treated with the following stimuli: vehicle control (DMEM), IL-1β 5 ng/mL, and Novobiocin at 100, 500 and 1000 μmol/L in presence or absence of IL-1β. Wells with serum-free medium were used as negative control. The cells were treated for 48 h. 3 h before each of the desired time points, 10 μl of the MTS reagent was added into each well and cells were further incubated at 37°C. The absorbance was detected at 490nm. All the experiments were repeated four times.
Western blotting was performed according to standard procedures. 20 μg of protein were loaded and resolved on standard 10% polyacrylamide SDS-PAGE gels. Separated proteins were then electroblotted onto polyvinylidene difluoride membranes (Millipore Co, Bedford, MA, USA). Equivalent loadings were verified by Ponceau Red staining after transference. Membranes were blocked in Tris-buffered saline, pH 7.4, containing 0.1% tween-20 (TBST), and 5% non-fat dried milk for 60 minutes at room temperature. The blots were then hybridized overnight at 4°C with antibodies against Hsp90β (1:1000), and α-tubulin (1:5000). All antibodies were diluted in TBST with 2% non-fat milk. After extensive washing with TBST, immunoreactive bands were detected by chemiluminiscence using the correspondent peroxidase-conjugated secondary antibodies and ECL detection reagents (GE Healthcare), and digitized using a LAS 3000 image analyzer. Quantitative changes of protein were evaluated with ImageQuant 5.2 software (GE Healthcare).
Induction and measurement of apoptosis
Apoptosis was induced by incubation of chondrocytes with sodium nitroprusside (SNP), or N-ethyl-2-(1-ethyl-2-hydroxy-2-nitrosohydrazine) ethanamine (NOC-12) at 0.5-1 mM in serum free DMEM for 24 hours. Cellular DNA content was assessed as described previously by flow cytometry . For this purpose, 2 × 105 cells were cultured in 6-well plates and treated as appropriate. Then, cells were spun and resuspended in a solution containing 1 mg/ml propidium iodide (PI) (Sigma) in PBS. Then, they were incubated at 4°C for 30 minutes in the dark and analyzed by flow cytometry on a FACSCalibur (BD Biosciences, San Jose, CA, USA) using a 560 nm dichromatic mirror and a 600 nm band pass filter. The percentage of cells with decreased DNA staining (composed of apoptotic cells resulting from either fragmentation or decreased chromatin) of a minimum of 10,000 cells per experimental condition was counted. The data are expressed as the percentage of hypodiploid (apoptotic) nuclei. Cells with a very low DNA content, in which the type of cell death could not be ascertained, were excluded from the analysis.
The apoptotic response was also measured in the silencing experiments by Cell Death Detection ELISAPLUS (enzyme-linked immunosorbent assay) (Roche Diagnostics, Mannheim, Germany, cat. N° 11774425001), following manufacturer's instructions and employing 2 × 104 cells seeded in 48-well plates. This kit is used for the quantitative in vitro determination of cytoplasmic histone-associated DNA fragments (mono- and oligonucleosomes) after induced cell death.
Transfection of small interfering RNA (siRNA)
For the silencing experiments we used a pre-plated transfection procedure. Approximately 72 hr before transfection, 8 × 104 healthy adherent cells were trypsinized and plated in 12-well plates, growing them in normal medium (DMEM, 10% FCS, 1% P/S, 1% gentamicin) until they reach 80% confluency after 48 hr. Then, medium was replaced with antibiotics-free DMEM. Cells were transfected 24 hours later, following manufacturer's instructions with minor modifications. Briefly, we used 7 μL/mL of siPORT Amine Transfection Agent (Ambion, cat. N° AM4502) and the final RNA concentration was 30 nM for HSPCB Silencer
Validated siRNA (Ambion, ref. AM51331). Controls were cells transfected without siRNA. Preliminary experiments determined that an incubation time of 72 h post-transfection was required to obtain an optimal level of HSPCB silencing. The transfected cells were treated with SNP and NOC-12, which served as apoptosis positive controls, and cell viability was assessed by trypan blue dye exclusion.
RNA isolation and real-time PCR assays
Total RNA was isolated from chondrocytes using Invisorb Mini Kit (Invitek, Berlin, Germany), following manufacturer's instructions. Whole RNA was treated with DNase (Invitrogen), and its concentration was determined by spectrophotometry. 1 μg of RNA from each sample was reverse-transcribed in a final volume of 20 μL using the Transcriptor First Strand cDNA Synthesis Kit (Roche Applied Science). cDNA synthesis was performed at 55°C for 30 minutes followed by a final step of 5 minutes at 85°C for inactivating the reverse transcriptase. Tubes were finally stored at -20°C until PCR analyses.
Primers for HSP90B and HPRT1 (housekeeping gene) were intron-spanning designed using the Universal Probe Library tool available at Roche website (http://www.roche-applied-science.com). Primer sequences were as follows: HSP90B forward, 5'-cgttgctcactattacgtataatcct-3'; HSP90B reverse, 5'-tgcctgaaaggcaaaagtct-3' (108 bp product); HPRT1 forward, 5'-tgaccttgatttattttgcatacc-3'; HPRT1 reverse, 5'-cgagcaagacgttcagtcct-3' (102 pb product).
Real-time PCR was performed in a LightCycler 480 instrument (Roche Applied Science), with 20 μL reactions containing 10 μL LightCycler 480 SYBR Green I Master, 7 μL bidistilled water, 0.5 μL (0.5 μM) each primer and 2 μL cDNA as PCR template. Cycling parameters were 95°C for 10 minutes to activate DNA polymerase, followed by 45 cycles of 95°C for 10 seconds, 60°C for 10 seconds and a final extension of 72°C for 10 seconds. Detection of fluorescence was carried out at the end of each extension step. After amplification, a melting curve was acquired by heating to 95°C for 5 seconds, cooling to 70°C for 1 minute and slowly heating to 95°C with a continuous fluorescence data collection of 10 acquisitions per°C.
PCR data were analyzed using REST (Relative Expression Software Tool) software, which provides statistical information suitable for comparing groups of treated versus untreated samples while taking into account issues of reaction efficiency and reference gene normalisation.
Chondrocytes were seeded at 5 × 104 cells per chamber in an 8-chamber slide. Cytokine stimulation was carried out on the chambers with IL1-β (5 ng/ml) or TNF-α (10 ng/ml) for 48 h. Then, media was removed and cells were fixed with acetone for 10 minutes at 4°C. After washing with PBS, anti-Hsp90β primary antibodies were applied at 1:50 dilution in PBS, and cells were incubated overnight in a humidified box at 4°C. After further washing, a FITC-conjugated anti-goat secondary antibody was used at 1:20 dilution for 1 h. Fluorescence microscopy was carried out in a Leica DMLS microscope. Quantification of the emitted fluorescence was performed with AnalySIS 5.0 software (Olympus Biosystems, Hamburg, Germany). Nuclei staining was performed with 4,6-dianidino-2-phenylindole dihydrochloride (DAPI, 2 mg/mL) for 30 minutes at 37°C.
Determination of nitric oxide levels by nitrite quantification
For the evaluation of nitric oxide production by chondrocytes, 5 × 104 cells were placed onto culture 96-well plates and allowed to adhere for 24 h. All conditions were set by duplicate. Nitric oxide production was stimulated by the addition of 5 ng/ml IL-1β, and the Hsp90 inhibitors Geldanamycin (GA) and Novobiocin (NB) (Alexis Biochemicals, Lausen, Switzerland) were added at 1, 10, 25 and 50 nM (GA), or 100, 500 and 1000 μM (NB). Then, supernatants were collected and total nitrite released in cell culture medium was measured by the Griess method , using sodium nitrite as standard. Data were expressed as μM nitrites (NO2
-) per number of cells per time.
The data are expressed as the mean (SEM) from n determinations or as representative results, as indicated. The statistical software program, SPSS, was used to perform the analysis of variance. Differences were considered to be significant at p < 0.05.