Antibodies to p38 MAPK, ATF-2, HSP27, NFκB-p65, IκB, phospho-p38 MAPK, phospho-ATF-2, phospho-HSP27, phospho-NFκB-p65 and phospho-IκB were obtained from Cell Signaling Technology (Beverly, MA) and Santa Cruz Biotechnology (Delaware, CA). The anti-human IL-8 antibody and biotin-conjugated anti-human IL-8 antibody were from BD PharMingen (San Diego, CA). The c-Src inhibitor PP2 and p38 MAPK inhibitor SB203580 were purchased from Calbiochem (La Jolla, CA).
Cell culture and viruses
Primary keratocytes were derived from donor corneas as previously described [30
]. Briefly, after mechanical debridement of the corneal epithelium and endothelium, corneas were cut into 2 mm-diameter sections, and placed in individual wells of six-well Falcon tissue culture plates with DMEM supplemented with 10% FBS, penicillin G sodium, and streptomycin sulfate at 37°C in 5% CO2
. Cells from multiple donors were pooled, and the cell monolayers used at passage three. For inhibitor analysis, cells were pretreated with PP2 (10 μM) or SB203580 (2, 5, 10 and 20 μM), for 3 hr at 37°C before infection. The cells were exposed to each inhibitor at the same concentrations throughout the infection process. Cell toxicity due to the inhibitors was ruled out by trypan blue exclusion performed on cells treated with inhibitors for the same time at the same concentrations. The protocol for use of corneas from deceased human donors was approved by the University of Oklahoma Institutional Review Board, and conformed to the tenets of the Declaration of Helsinki.
HAdV-19 used in this study was isolated from a human patient's cornea, as previously described [28
], and grown in A549 cells, (lung carcinoma cells, CCL 185; American Type Culture Collection, Manassas, VA) in MEM with 2% FBS, penicillin G sodium, and streptomycin sulfate. The Oklahoma State Department of Health confirmed the viral serotype. Virus was purified from A549 cells by ultracentrifugation via CsCl gradient, dialyzed against a 10 mM Tris (pH 8.0) buffer that contained 80 mM NaCl, 2 mM MgCl2
, and 10% glycerol, titered in triplicate and stored at -80°C.
Monolayer cells grown to 95% confluence in six-well plates were washed in MEM with 2% FBS, and infected with purified HAdV-19 at a multiplicity of infection (MOI) of 50 or mock infected with virus-free dialysis buffer as a control. Virus was adsorbed at 37°C for 1 hr and then incubated for 1 additional hr before RNA isolation. For protein analysis, cells grown to 95% confluence in six-well plates were serum-starved for 18–24 hr before infection, and lysed at 4 hours post-infection.
HAdV-19 and mock infected keratocytes were lysed with chilled cell lysis buffer (20 mM Tris, pH 7.4, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% Triton X-100, 2.5 mM sodium pyrophosphate, 1 mM β-Glycerolphosphate, 1 mM Na3VO4, 1 μg/ml Leupeptin, and 1 mM PMSF), and incubated at 4°C for 5 min. The cell lysates were cleared by centrifugation at 21,000 × g for 15 min. The protein concentration of each supernatant was measured by BCA analysis (Pierce, Rockford, IL) and equalized. Twenty micrograms of cell lysates were subsequently separated by 10% SDS-PAGE and transferred onto nitrocellulose membranes (BioRad, Hercules, CA) and immunoblotted. The bands were visualized with an enhanced chemiluminescence kit (Amersham, Piscataway, NJ). Densitometric analysis of immunoblots where indicated was performed using ImageQuant 5.2 (Amersham) in the linear range of detection, and absolute values were then normalized to total protein or actin as indicated in figure legends.
p38 MAPK assay
p38 MAPK activity was determined using the p38 MAPK Assay Kit (Cell Signaling). Briefly, endogenous p38 MAPK was immunoprecipitated from 250 μg of cell lysate with immobilized phospho-p38 MAPK (Thr180/Tyr182) monoclonal antibody overnight at 4°C. The precipitates were washed twice with lysis buffer and twice with kinase buffer (25 mM Tris, pH 7.5, 5 mM β-glycerophosphate, 2 mM DTT, 0.1 mM Na3VO4, and 10 mM MgCl2), and suspended in kinase buffer containing cold ATP (200 μM) and ATF-2 fusion protein. After incubation for 30 min at 30°C, the reactions were stopped with 3 × SDS sample buffer (187.5 mM Tris-HCl pH 6.8, 6% w/v SDS, 30% glycerol, 150 mM DTT, 0.03% w/v bromophenol blue). The proteins were resolved by 10% SDS-PAGE followed by western blot analysis. The membranes were probed with antibodies against phospho-ATF-2 (Thr76). Phosphorylated ATF-2 protein from three different experiments was quantified using densitometer scanning, and the means compared by Student's t test for each time point.
Total RNA was isolated using TRIzol reagent (Invitrogen, Carlsbad, CA) according to the manufacturer's protocol. RNA concentrations and quality were determined spectrophotometrically. The template, cDNA was synthesized by reverse transcription of the total RNA (2 μg) with Moloney murine leukemia virus reverse transcriptase (Promega, Madison, WI) using an oligo(dT) 15 primer (Promega). The primers used for PCR amplification included: IL-8 (Genbank #: AF385628) forward, 5'GTGTGGGTCTGTTGTAGGGT3'; reverse, 5'CTGTGAGGTAAGATGGTGGC3', which amplified a 481-bp product; GAPDH (Genbank #: X01677) forward, 5'GTCGGAGTCAACGGATTTGGTCGT3'; and reverse, 5'GACGGTGCCATGGAATTTGCCATG3', which yielded a 165-bp product. The PCR reaction was performed on Mastercycler® (Eppendorf, Hamburg, Germany) using the following cycling parameters: 94°C for 2 min, 30 cycles of 94°C for 15 sec, 55°C for 15 sec and 72°C for 45 sec, followed by the final step of 72°C for 1 min. The amplification products were analyzed by gel electrophoresis on 1% agarose gel.
Whole-cell lysates from infected primary keratocytes (300 μg) were precleared with protein A-Sepharose beads for 30 min. Precleared protein extracts were added to anti-p38 MAPK (Santa Cruz Biotechnology) or isotype control (anti-rabbit) antibodies in phosphate-buffered saline containing protease inhibitors (phenylmethylsulfonyl fluoride [5 × 10-5 M], leupeptin [1 × 10-2 mg/ml], aprotinin [5 × 10-3 mg/ml], and sodium vanadate [30 mM]), 0.1% Tween 20 and rocked at 4°C for 2 h before the addition of protein A-Sepharose (25 μl; 1:1 slurry) and further incubated at 4°C for 12 h. Immunoprecipitates were washed five times with wash buffer (100 mM Tris-Cl pH 8.0, 500 mM NaCl, 0.1% Tween 20) containing protease inhibitors, and proteins were eluted by the addition of sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis sample buffer and boiling for 5 min. Samples were run on 10% SDS-polyacrylamide gels using standard protocols and transferred to nitrocellulose membranes (BioRad). The membrane was probed with anti-phospho-NFκB-p65 and the bands were visualized with an enhanced chemiluminescence kit (Amersham).
Electrophoretic mobility gel shift assay
Nuclear extracts from HAdV-19 infected and buffer treated (mock) keratocytes were prepared using Nucbuster kit (Novagen, Madison, WI). Binding and supershift assays were done using Dig Gel Shift kit (Roche, Indianapolis, IN) according to the manufacturer's instructions. Briefly, IL-8 sense and anti-sense oligos encoding specific binding sites for NFκB were synthesized (IDT, Coralville, IA) and annealed. Oligos were then labeled using Dig-ddUTP and terminal transferase for 15 min at 37°C in the labeling buffer. For the assay, 5 μg of nuclear extract, labeled oligo-nucleotide, poly (dI-dC) (1 μg), and poly L-lysine (0.1 μg) were mixed in the binding buffer and incubated at room temperature for 15 min. For competition, 100 molar excess of unlabelled probe was added to the reactions 15 min before the addition of labeled probe. For supershift assay, 1 or 2 μg of NFκB-p65 antibody was added to binding reaction and incubated on ice for 30 min prior to adding the probe. Protein-DNA complexes were resolved in 5% pre-electrophoresed polyacrylamide gel in 0.5× TBE running buffer and then transferred to a nylon membrane (Roche). The membrane was then probed for anti-digoxigenin and the bands were detected by chemiluminescense using a Kodak Image Station 4000R (Rochester, NY).
Keratocytes grown on slide chambers (Nunc, Rochester, NY) were treated with DMSO or SB203580 (10 μM or 20 μM) for 3 hr and then infected with HAdV-19 or dialysis buffer for 20 min. Cells were partially fixed in 0.05% paraformaldehyde for 10 min, washed in PBS containing 2% FBS, and permeabilized in solution containing 0.1% Triton X-100 for 5 min. After 30 min blocking in 3% FBS-PBS, the cells were incubated in 5 μg/ml of NFκB-p65 primary antibody for 1 hr at room temperature, washed and incubated in Alexafluor-594 conjugated secondary antibody (Molecular Probes, Eugene, OR) for 1 hr more at room temperature. Cells were then washed, fixed in 2% paraformaldehyde, and mounted using Vectashield (Vector labs, Burlingame, CA) mounting medium containing DAPI. Images were taken in an Olympus (Center Valley, PA) FlouView 500 confocal microscope using a 60× water immersion objective.
Keratocytes were treated with DMSO or SB203580 (2, 5, 10, and 20 μM) for 3 hr before infection with purified HAdV-19 or virus-free dialysis buffer as a control. The cell supernatants were collected 4 hr post-infection, and the levels of IL-8 quantified by sandwich ELISA. The detection limit was 30 pg/ml. Plates were read on a SpectraMax M2 microplate reader (Molecular Devices, Sunnyvale, CA) and analyzed with SOFTmax analysis software (Molecular Devices). The means of triplicate ELISA values for each of the virus- and mock infected wells were determined, and a dose-response relationship between p38 MAPK inhibitor concentration and IL-8 protein expression examined by linear regression analysis.
Transfections were carried out using Oligofectamine (Invitrogen) following the manufacturer's instructions. Briefly, the transfection mixture was prepared by mixing 12 μl of Oligofectamine to 48 μl of Opti-MEM followed by incubation at room temperature for 5 min, followed by addition of 100 nM SMARTpool p38-MAPK siRNA or control siRNA (Upstate, Charlottesville, VA) and further incubation for 15 min. The transfection complex was added to 50–70% confluence cells, and virus and mock infections carried out 48 hours later. Supernatants and cell lysates were collected 4 hour after infection for IL-8 ELISA and p38 MAPK western blot analysis, respectively. The effect of siRNA on IL-8 ELISA was determined by ANOVA with preplanned contrasts.