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Role of survivin in acute lung injury: epithelial cells of mice and humans
Yasuhiro Terasaki1, Mika Terasaki1, Hirokazu Urushiyama1, Shinya Nagasaka1,
Mikiko Takahashi1, Shinobu Kunugi1, Arimi Ishikawa1, Kyoko Wakamatsu1, Naomi
Kuwahara1, Koichi Miyake2, and Yuh Fukuda1
MATERIALS AND METHODS
Antibodies
Antibodies for survivin included AF886 (1:1000; R&D Systems, Wiesbaden,
Germany); surfactant protein-C (SP-C) (Santa Cruz Biotechnology, Santa Cruz, CA,
USA); surfactant protein A (SP-A) (Dako, Glostrup, Denmark); S100A4 (Acris
Antibodies, Inc. San Diego, CA, USA); F4/80 (Serotec, Oxford, UK); CD68 (Dako);
Smac/DIABLO, activated caspase-3, and activated poly (ADP-ribose) polymerase
(PARP) (Abcam Inc., Cambridge, MA, USA); caspase-3, activated caspase-7,
caspase-7, and PARP (Cell Signaling Technology, Inc., Danvers, MA, USA);
GAPDH (Epitomics, Inc., Burlingame, CA, USA); and PCNA (Dako).
Bleomycin-Induced Lung Injury Mouse Model
Pulmonary fibrosis was induced in 8-wk-old male ICR mice (weight 35-40 g;
Sankyo Labo Service Corporation, Inc., Tokyo, Japan) by one intratracheal
instillation of bleomycin hydrochloride (25 mg/kg body weight; Nippon Kayaku Co.,
Tokyo, Japan), administered via a 27-gauge needle placed between the cartilaginous
tracheal rings, and with the animals under i.p. induced pentobarbital sodium
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anesthesia. Control animals received injections of the same volume of saline.
The
Animal Care and Use Committee of Nippon Medical School approved the animal
protocols.
The mice were housed five per cage and kept under standard laboratory
conditions, with food and water supplied ad libitum.
On specific days after the
bleomycin injection (days 0, 3, 7, and 14), eight bleomycin-treated and five control
animals were anesthetized with an i.p. injection of 40 mg/kg pentobarbital sodium
and were exsanguinated by means of cutting the abdominal aorta. The tracheas
were cannulated, and lungs were removed. The left lungs were immediately frozen
at −80 °C and were used later for protein and RNA extraction. The right lungs,
which were used for microscopy, underwent an 8-h fixation at 4 °C in 4%
paraformaldehyde in 0.1 M phosphate buffer (pH 7.4) and were inflated at a pressure
of 20 cm H2O. They were embedded in paraffin or sequentially washed in
10%, 15%, and 20% sucrose in 0.01 M phosphate-buffered saline (PBS; pH 7.4, 4°C)
for 4 h, washed with 20% sucrose in 0.01 M PBS overnight, and then snap-frozen in
O.C.T. embedding medium and stored at −80 °C.
Patients
Lung specimens were obtained via autopsies at the Nippon Medical School Hospital
during 1992–2011: 10 DAD autopsy cases, 8 autopsy control cases.
Cases with
DAD that was due to other or complicated causes were excluded. For the autopsy
cases used in the present study, the postmortem time was less than 5 h.
All
specimens were diagnosed by using American Thoracic Society/European
Respiratory Society International Multidisciplinary Consensus (1).
Ages in the
DAD group ranged from 34 to 81 years (mean age, 68.1 years; male/female ratio =
2
5:5), and those in the normal control group, from 48 to 87 years (mean age, 71.5
years; male/female ratio = 5:3) (Table 1). Cases of antecedent interstitial
pneumonia associated with DAD changes were not included. To confirm the
histological diagnosis and morphological evaluations, three pathologists reviewed
the slides.
The study protocol was approved by the Human Ethics Review
Committee of Nippon Medical School.
Written informed consent was obtained
from families of patients.
Immunohistochemistry and Evaluation for Survivin-Positive Cells
Paraffin-embedded mouse and human lung sections were prepared serially, with
some as mirror images.
The sections were deparaffinized and then microwaved in
DakoCytomation Target Retrieval Solution (Dako) for 10 min at 100 C, followed by
slow cooling and washing in PBS three times. After endogenous peroxidase
activity and nonspecific binding sites were blocked with Peroxidase Blocking
Reagent (Dako) and 5% normal goat or rabbit serum (Vector Laboratories, Inc.,
Burlingame, CA, USA) in PBS, sections were incubated overnight at 4 C with the
indicated primary antibodies. After slides were washed, they were incubated with
horseradish peroxidase (HRP)-conjugated anti-rabbit antibodies (LSAB Kit, Dako)
or HRP-conjugated anti-goat antibodies (Histofine Simple Stain MAX PO (G);
Nichirei Biosciences Inc., Tokyo, Japan) or HRP-conjugated anti-mouse antibodies
with Blocking Reagent A and B (Histofine Mousestain Kit; Nichirei Biosciences
Inc.) for 1 h at room temperature. Negative controls included normal rabbit IgG
and mouse IgG.
Bound peroxidase was detected with 3,3-diaminobenzidine
tetrahydrochloride (DAB; Dojindo Laboratories, Kumamoto, Japan) and H2O2.
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Slides were counterstained with Mayer’s hematoxylin (Merck, Darmstadt, Germany).
The ratio of the number of bronchial epithelial cells with positive results for survivin
in the nucleus or cytoplasm to the total number of bronchial epithelial cells was
determined in blind fashion for six fields (three from the upper lobe and three from
the lower lobe) for all groups of mice (n = 4). The numbers of alveolar epithelial
cells with positive results for survivin in the nucleus or cytoplasm, per high-power
field, were counted in blind fashion for six fields for all groups of mice (n = 4). The
numbers of alveolar epithelial cells with positive results for survivin in the nucleus or
cytoplasm, per high-power field, were counted in blind fashion for six fields (three
from the upper lobe and three from the lower lobe) for lungs from DAD cases and
control subjects (n = 10 and n = 8, respectively).
Terminal Deoxynucleotidyl Transferase (TdT) dUTP Nick End Labeling
(TUNEL)
For in situ analysis of DNA fragmentation, TUNEL staining was performed with a
commercial kit (ApopTag Plus Peroxidase In Situ Apoptosis Detection Kit S7101;
Chemicon, Temecula, CA), according to the manufacturer’s instructions.
In brief,
paraffin-embedded lung sections from mice were deparaffinized, were washed twice
with PBS, and were digested with proteinase K (20 μg/ml) for 7 min, and then 3%
H2O2 was used for 20 min to quench endogenous peroxidase. After sections were
washed twice with PBS, they were incubated with TdT for 60 min at 37 °C. The
sections were then incubated with anti-digoxigenin-peroxidase for 60 min at room
temperature, followed by color development with H2O2-DAB for 20 min and then
three washes in Milli-Q water. Mayer’s hematoxylin was used for counterstaining,
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and sections were covered with coverslips. For the technical negative control, TdT
was omitted from the incubation buffers during processing.
Double Labeling for Immunohistochemistry and TUNEL Staining
After endogenous peroxidase activity and nonspecific binding sites were blocked,
slides were incubated overnight with the first primary antibody against survivin,
followed by incubation with HRP-conjugated anti-rabbit antibodies (Histofine
Simple Stain MAX PO (R) ; Nichirei Biosciences Inc.) for 1 h and visualization with
aminoethylcarbazole (AEC; red chromogen) substrate solution (Histofine Simple
Stain AEC Solution; Nichirei Biosciences Inc.).
The first primary antibody against
survivin was inactivated by boiling in 10 mM citrate buffer (pH 6.0) for 10 min to
prevent mixed labeling and cross-reaction (2). Slides were incubated overnight
with the second primary antibodies against SP-C and S100A4, followed by
incubation with alkaline phosphatase (AP)-conjugated anti-goat antibody (Histofine
Simple Stain AP (G); Nichirei Biosciences Inc.) for 1 h and visualization with AP
substrate solution (Vector Blue Alkaline Phosphatase Substrate Kit; Vector
Laboratories, Inc., Burlingame, CA, USA).
After endogenous peroxidase activity and nonspecific binding sites were blocked,
slides were incubated overnight with the first primary antibody against PCNA,
followed by incubation with HRP-conjugated anti-mouse antibodies (Mousestain Kit
PO (M); Nichirei Biosciences Inc.) for 1 h and visualization with DAB (brown
chromogen). The first primary antibody was inactivated by boiling in 10 mM
citrate buffer (pH 6.0) for 10 min to prevent mixed labeling and cross-reaction (2).
Slides were incubated overnight with the second primary antibody against survivin,
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followed by incubation with AP-conjugated anti-rabbit antibody (Histofine Simple
Stain AP (R); Nichirei Biosciences Inc.) for 1 h and visualization with AP substrate
solution (New Fuchsin Substrate kit; Nichirei Biosciences Inc.).
After TUNEL staining without Mayer’s hematoxylin counterstaining,
immunohistochemistry for suvivin was performed, with incubation overnight with
survivin antibody followed by incubation with HRP-conjugated anti-rabbit
antibodies (Histofine Simple Stain MAX PO (R); Nichirei Biosciences Inc.) for 1 h
and visualization with AEC (red chromogen) substrate solution (Histofine Simple
Stain AEC Solution; Nichirei Biosciences Inc.).
Immunoelectron Microscopy
Frozen mouse lung tissues fixed with 4% paraformaldehyde were cut into 6-µm
sections, which were treated with Peroxidase Blocking Reagent (Dako) and 5%
normal goat serum (Vector Laboratories) in PBS for 30 min and were then incubated
with anti-survivin antibody overnight at 4 °C, followed by a 90-min incubation with
HRP-conjugated goat-anti-rabbit IgG F(ab)2 (Biosource International, Camarillo,
CA, USA). The sections were then fixed with 1% glutaraldehyde for an additional
5 min, reacted with DAB and H2O2, postfixed in 2% osmium tetroxide for 60 min,
dehydrated in a graded series of ethanol, and embedded in Epon 812 resin.
The
ultrathin sections were viewed with a transmission electron microscope (Hitachi
H-7500; Hitachi Ltd., Tokyo, Japan) at 75 kV.
Real-time Reverse Transcription-Quantitative Polymerase Chain Reaction
(RT-qPCR) Amplification
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Survivin mRNA expression was analyzed by performing real-time PCR. Total
RNA was extracted with TRIzol reagent (Qiagen, GmbH, Hilden, Germany)
according to the manufacturer's instructions.
Poly A+ RNA was treated with DNase
I (RNase-Free DNase Set, Qiagen) to remove contaminating DNA. The OD
260/280 nm ratios of all samples were >1.8.
To generate first-strand cDNA, total
RNA (100 ng) was reverse-transcribed with the high-capacity cDNA Reverse
Transcription Kit (Applied Biosystems, Foster City, CA) in a total volume of 20 μl.
In each PCR reaction, 10 μl of cDNA was used (total volume 25 μl).
Ready-to-use
primer and probe sets from Applied Biosystems, Foster City, CA, USA.
(Assay-on-Demand Gene Expression Catalog number Mm00599749_m1, and
GAPDH) were used to detect mouse survivin mRNA. Primer and probe
concentrations for each target gene were optimized according to the manufacturer's
procedure. PCR (2 min at 50 °C, 10 min at 95 °C, and 45 cycles of 15 s of
denaturation at 95 °C and 60 s of annealing at 60 °C) was performed on the ABI
Prism 7000 Sequence Detection System (Applied Biosystems) with the fluorescent
TaqMan methodology.
tRNA from one lung homogenate was used as a standard
sample (SS) for survivin mRNA and GAPDH mRNA. To create a standard curve,
10-fold dilutions of the SS were used, i.e., 3, 0.3, 0.03, 0.003, 0.0003, and
none. For all experiments, survivin mRNA and GAPDH mRNA were quantified in
triplicate and survivin mRNA was normalized against GAPDH mRNA. Results are
expressed relative to the SS (1  SS = 1.0).
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Cell Culture
BEAS-2B human bronchial epithelial cells were obtained from American Type
Culture Collection (Manassas, VA, USA) and were grown in culture dishes or
culture slides coated with type VI collagen (collagen from human placenta;
Sigma-Aldrich, Inc., St. Louis, MO, USA) with bronchial epithelial cell basal
medium as the maintenance medium (Lonza Walkersville, Inc., Walkersville, MD,
USA) and with growth supplements (bovine pituitary extract, 2 ml; hydrocortisone,
0.5 ml; human epidermal growth factor, 0.5 ml; epinephrine, 0.5 ml; transferrin, 0.5
ml; insulin, 0.5 ml; retinoic acid, 0.5 ml; triiodothyronine, 0.5 ml; GA-1000, 0.5 ml;
Lonza Walkersville, Inc.) in a 5% CO2 humidified chamber. Human lung alveolar
epithelial cell line A549 cells were obtained from the Cell Research Center for
Biomedical Research (Institute of Development, Aging and Cancer, Tohoku
University) and were grown in culture dishes or culture slides with RPMI as the
maintenance medium (Life Technologies Corporation, Carlsbad, CA, USA)
containing 10% fetal bovine serum and 1% penicillin-streptomycin (Life
Technologies Corporation) in a 5% CO2 humidified chamber.
Preparation of Whole-Cell Lysates
Cells were incubated for 24 h in 6-well dishes in complete medium to allow
attachment before replacement of the medium with serum-free medium.
After
incubation for 24 h without serum, cells were treated with bleomycin as indicated.
After cells were rinsed with Dulbecco’s PBS (Invitrogen, Carlsbad, CA, USA), they
were homogenized in mammalian protein extraction reagent containing Protein
Stabilizing Cocktail, Halt Protease Inhibitor Cocktail, Halt Phosphatase Inhibitor
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Cocktail (Thermo Scientific, Rockford, IL, USA), 150 mM NaCl, and 1 mM EDTA.
Lysates were centrifuged (12,000 rpm, 5 min), and the supernatant was termed
“whole-cell lysate.” Protein concentration was measured by means of the Bradford
method.
Western Blotting
For each experiment, Western blotting was performed according to a standard
procedure.
Lysates including equal amounts of protein were boiled in sodium
dodecyl sulfate (SDS) sample buffer for 5 min, after which they were separated with
10% SDS-polyacrylamide gel electrophoresis and then transferred with an
electroblot apparatus (Invitrogen) to polyvinylidene difluoride membranes
(Invitrogen). Membranes were incubated with protein-free T20 Tris-buffered saline
(TBS) blocking buffer (Thermo Scientific) for 1 h at room temperature, and then
they were incubated with antibodies, diluted 1:1000, against survivin, activated
PARP, PARP, activated caspase-3, caspase-3, activated caspase-7, caspase-7, or
GAPDH at 4 °C for about 16 h.
After TBS containing 0.1% Tween 20 (TBS-T)
was used to wash membranes several times, the membranes were incubated for 45
min with the proper HRP-conjugated second antibodies (Promega, Madison, WI,
USA), washed with TBS-T, and developed with SuperSignal West Femto
Luminol/Enhancer Solution (Thermo Scientific, Rockford, IL, USA).
Immunoreactivity was detected via the LAS-4000 Luminescent Image Analyzer and
a CCD Camera (Fujifilm, Tokyo, Japan) and was quantified via densitometry and
Fuji Image Gauge software (version 4.0; Fujifilm). The proteins were stripped from
the blotting membrane by a 5-min incubation in Restore PLUS Western Blot
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stripping buffer (Thermo Scientific). After reactions for GAPDH protein, each
protein was quantified, expressed as a ratio to the amount of GAPDH protein.
Five
respective results are reported relative to corresponding results for controls (no
treatment) in five experiments (no treatment = 1.0).
Detection of Cell Damage
Culture medium, collected after incubation of cells under the indicated conditions,
was analyzed for lactate dehydrogenase release (an indicator of cell damage) by
using the Cytotoxicity Detection KitPLUS (Roche Applied Science, Mannheim,
Germany) according to the manufacturer’s instructions. Optical density (OD) was
measured at 490 nm with a Model 550 Microplate Reader (Bio-Rad Laboratories Inc.,
Hercules, CA, USA). A sample OD was quantified as the experimental OD490 −
spontaneous OD490.
The experimental OD value was determined for the serum-free
culture medium of cells cultured under the indicated conditions.
The spontaneous
OD value was determined for the serum-free culture medium (e.g., bronchial
epithelial cell basal medium). Five OD results were reported relative to
corresponding results for controls (no treatment) in five experiments (control
treatment = 1.0).
We also assessed cell survival by manually counting cells that were double-stained
with Annexin V-FITC (BD Biosciences, San Jose, CA, USA; cell membranes of
apoptotic cells stained green) and 1 mM propidium iodide (PI) (Invitrogen; nuclei of
dead cells stained pink) as well as 5 mM Hoechst 33342 dye (Invitrogen; nuclei of
dead and living cells stained blue). The ratios Annexin V/Hoechst-positive and
PI/Hoechst-positive cells obtained by manual counting were determined in blind
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fashion for five fields (n = 4).
Small Interfering RNA (siRNA) Experiments
siRNA targeting human survivin and nontargeting control siRNA were obtained from
Sigma-Aldrich (MISSION siRNA and MISSION siRNA Universal Negative Control,
respectively).
Transfection was performed with Lipofectamine RNAiMAX reagent
(Invitrogen), according to manufacturer’s protocol, in 1 ml of Opti-MEM I Reduced
Serum Medium (Invitrogen).
Survivin Overexpression Studies
pcDNA3HA-survivin, a survivin expression vector with an N-HA tag, was purchased
from CH3 BioSystems, LLC (Amherst, NY, USA).
Cells were plated on 35-mm
dishes at a density of 2.3  105 cm2 for 24 h before transfection. After 24 h, cells
were transfected with Lipofectamine 2000 (Invitrogen) and FuGENE HD
Transfection Reagent (Roche Applied Science) according to the manufacturers’
instructions.
Empty vector transfection (pcDNA3; Invitrogen) served as a negative
control. The transfection medium was removed and replaced with full growth
medium at 6 h after the transfection.
Transfection efficiency was controlled by
immunoblotting.
Statistical Analysis
For each data set, arithmetic means and SEM values were calculated; Student’s t-test
was used to compare paired or independent variables.
to determine statistical differences among groups.
One-way ANOVA was used
*P < 0.05 and **P < 0.01 were
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considered statistically significant.
References
1
American Thoracic Society/European Respiratory Society International
Multidisciplinary Consensus Classification of the Idiopathic Interstitial
Pneumonias.
2
Am J Respir Crit Care Med 2002;165:277-304.
Ikeda K ST, Tate G, Mitsuya T. Multiple immunoenzyme labeling using heat
treatment combined with the polymer method: an analysis of the appropriate
inactivation conditions of primary antibodies. Acta Histochem
2011;113:117-124.
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