Manuscript # CVR-2011-1297R3
Original Research
Aquaporin 1, Nox1 and Ask1 Mediate Oxidant-Induced Smooth Muscle Cell Hypertrophy
Imad Al Ghouleh1,2, Giovanna Frazziano1,2,7, Andres I Rodrigues1,2, Gabor Csanyi1,2, Salony Maniar3,
Claudette M St Croix3,4, Eric E Kelley1,2,5, Loreto A Egana1,2 , Gyun Jee Song2, Alessandro Bisello2,
Yong J Lee2,6, and Patrick J Pagano1,2,*
Running title: Aqp1, Nox1 and Ask1 Mediate Smooth Muscle Hypertrophy
Detailed Materials and Methods:
Materials
Hydrogen peroxide (H2O2), cytochrome c, collagenase, elastase, diphenylene iodonium (DPI),
rotenone, 4,5-Dihydroxy-1,3-benzene-disulfonic acid (Tiron), N-Nitro-L-arginine methyl ester
hydrochloride (L-NAME), superoxide dismutase (SOD), and indomethacin were purchased from
Sigma (Sigma-Aldrich, St. Louis, MO). Febuxostat was purchased from Axon Medchem (Groningen,
The Netherlands). L-012 was purchase from Wako Chemicals (Wako Chemicals USA Inc.,
Richmond, VA). 1-hydroxy-3-methoxycarbonyl-2,2,5,5-tetramethylpyrrolidine hydrochloride (CMH)
and deferoxamine were purchased from Noxygen Science Transfer (Germany). HyPer plasmid was a
kind gift from Dr. David Pimental (Boston Medical Center, Boston, MA). Trypsin and DMEM were
purchased from Mediatech (Mediatech Inc., Manassas, VA). Optimem, Lipofectamine 2000, Nox1,
Aqp1 and scrambled Stealth siRNA, and fetal bovine serum (FBS) were purchased from Invitrogen
(Carlsbad, CA). Rabbit anti-total Ask1 antibody was purchased from Cell Signaling Technology
(Boston, MA). Rabbit anti-phospho Ask1 (threonine-845) was a kind gift from Dr. Hidenori Ichijo
(The University of Tokyo, Japan).
O2•– Detection by Electron paramagnetic resonance (EPR)
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The EPR spin probe CMH was used to examine O2•– production using a Bruker eScan TableTop EPR spectrometer (Bruker Biospin, USA). Cells were trypsinized and resuspended in KrebsHEPES buffer (pH 7.4) at a concentration of 1 x 106 cells/ml. O2•– production was measured by
adding CMH (50 μM) to the cell suspension followed by 50 µM H2O2 or PBS (vehicle). Suspensions
in the presence or absence of SOD (200 U/ml) were transferred to an EPR capillary tube. Spectra
were obtained after 1, 10 and 20 minutes 37C. To minimize the deleterious effects of contaminating
metals, the buffers were treated with Chelex resin and contained 25 μM of the iron chelator
deferoxamine.
O2•– Detection by L-012 chemiluminescence
Cells were grown to 90% confluence on white clear-bottom 96-well culture plates and serum
starved for 18-24 hours. Cells were stimulated with varying concentrations of H2O2 for 1 hr (or 10 –
300 min for time-course experiments). The media was then replaced with PBS (pH 7.4) supplemented
with CaCl2 (0.9 mM) and MgSO4 (0.49 mM) and luminol derivative L-012 (400 µM) was added
followed by chemiluminescence measurement in BioTek Synergy 4 Hybrid Multi-Mode Microplate
Reader (BioTek, Winooski, VT) for at least 30 min post the 1 hr H2O2 stimulation. In some
experiments, cells were pre-incubated for 30 min with pharmacological inhibitors prior to addition of
H2O2. In other experiments, SOD (300 U/ml) was added immediately prior to L-012 additions. O2•–
production was quantified as relative light units (RLU).
O2•– Detection by Cytochrome c Reduction Assay
Cytochrome c reduction assay was conducted as described previously
20
. Briefly, cells were
grown to 90% confluence, serum starved and treated with 50 M H2O2 for 1 hr. Cells were washed
twice with ice cold PBS and 400 l of ice cold disruption buffer (8 mM potassium, sodium phosphate
buffer, pH 7.0, 131 mM NaCl, 340 mM sucrose, 2 mM NaN3, 5 mM MgCl2, 1 mM EGTA, 1 mM
EDTA, and protease inhibitor cocktail) was added. Cells were then scraped and subjected to 5
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freeze/thaw cycles followed by 5 passages through a 30-guage needle to lyse them. Lysates were
centrifuged at 1000 g for 10 min at 4°C and the supernatant collected and further centrifuged at
28000g for 20 min at 4°C to collect the membrane fraction pellet, which was resuspended in 100 l
disruption buffer. Membrane fractions (5 – 10 g/well) were added to cytochrome c-containing
oxidase assay buffer (65 mM sodium phosphate buffer, pH 7.0, 1 mM EGTA, 10 μM FAD, 1 mM
MgCl2, 2 mM NaN3, and 0.2 mM cytochrome c) and O2•– measured as the initial rate of SODinhibitable cytochrome c reduction quantified at 550 nm using the extinction coefficient 21.1 mM− 1
cm− 1. After a 5 min baseline measurement, NADPH (180 M) was added and O2•– production was
calculated from the initial linear rate (first 10 min) of cytochrome c reduction.
Live-cell Imaging Confocal Microscopy of HyPer Fluorescence
Cells were grown to 50% confluence on coverslip glass bottomed (#1.5) petri dishes (MatTek
Corp., Ashland, MA) and transfected with HyPer plasmid using the transfection reagent
Lipofectamine 2000 according to the manufacturer’s protocol. 24 – 48 hr later, media was changed to
Optimem and plates were mounted in a temperature controlled chamber (Tokai Hit Co., Shizuokaken, Japan) atop the motorized stage of a Nikon TiE inverted fluorescent microscope equipped with a
60X, 1.4 NA optic (Nikon Inc., Melville, NY). HyPer was excited by the 438 nm and 513 nm lines of
a SpectraX light engine (Lumencor Inc., Beaverton OR) and detected using a 525/50 nm bandpass
filter (Chroma Technology Corp), Hamamatsu C11440-22C camera (Hamamatsu Corp.), and NIS
Elements software. The ratios (513/438) were calculated for 1-3 cells per stage position with 10 stage
positions per plate per experiment. After 5 minutes of imaging, 50 µM H2O2 was added to the cells
and imaging resumed for an additional 20 – 60 min.
Adenoviral Transduction for Ask1 Gain- and Loss-of-Function
Cells were grown to 50% confluence and the media changed for no serum conditions.
Adenoviruses for GFP, Transgenic (Tg)- Ask1 or dominant negative (DN)- Ask1 (a generous gift
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from Drs. Yong Lee, University of Pittsburgh,
and Jae J Song, Yonsei University College of
Medicine, Seoul, Republic of Korea) were added to cells at a multiplicity of infection (MOI) of 10
and incubated for 24 hr at 37C. Cells were washed and growth media added for 6 or 24 hr followed
by serum starvation and treatment. For experiments in which siRNA was applied, cells were
transduced with Ask1 adenoviruses 24 hr post siRNA transfection.
Quantitative PCR
Cells were lysed in RLT buffer and RNA was purified using RNeasy Plus Mini Kit (Qiagen). 1-2 µg
of total RNA was used to prepare cDNA using SuperScript™ First-Strand Synthesis System
(Invitrogen) as per the manufacturer’s protocol and qPCR was performed using the TaqMan®
Universal PCR Master Mix (ABI- Applied Biosystems Inc, Foster City, CA) according to the
manufacturer’s protocol. Samples were mixed with primer/probe mixes for Nox1, Nox4, Aqp1 or
18S (ABI) in 384-well qPCR plates (ABI) and amplification/measurement was obtained in a 7900HT
Fast Real-Time PCR System (ABI) according to the manufacturer’s protocol for 40 cycles. Relative
quantification was obtained using the Ct (threshold cycle) method:
Ct = CtNox1/Nox4/Aqp1 – Ct18S; Ct = CtNox1/4/Aqp1 siRNA transfected sample - CtScrambled siRNA transfected sample
Relative expression was calculated as 2-ΔΔCt.
Western Blot
Following stimulation, cells were washed with ice cold PBS (1x) and lysed with RIPA buffer
supplemented with protease and phosphatase inhibitors (Roche Diagnostics GmbH, Mannheim,
Germany). Cells were centrifuged at 1000 × g for 10 min at 4C, and the supernatant collected.
Protein concentration was measured using the Bradford method (Thermo Scientific, Rockford, IL).
Samples were prepared with Tris-Glycine SDS sample buffer, resolved by SDS–PAGE along with a
molecular weight standard (Bio-Rad Laboratories, Hercules, CA), and transferred onto Trans Blot
nitrocellulose membranes (Bio-Rad). Membranes were blocked with the Odyssey Blocking Buffer
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(Li-Cor Biosciences, Lincoln, NE) and incubated with total Ask1 or phospho-Ask1 antibodies (1:500
dilution). Membranes were probed with goat anti-mouse or goat anti-rabbit secondary antibodies
(1:10 000 dilution, Li-Cor Biosciences). Digital imaging was obtained using the Odyssey Infra-Red
Imaging system (Li-Cor Biosciences).
Quantification of Cell Hypertrophy
rASMC were grown to 80% confluence then serum starved as indicated above and treated
with 50 M H2O2 for 24 hr 37°C. Cells were then trypsinized, resuspended in 1 ml ice cold PBS,
separated into two equal volumes (500 L each) and centrifuged at 1000 g for 10 min at 4°C. One
volume of cells was lysed in RIPA buffer as in Western Blot above for protein content quantification
and the other volume was resuspended in DNAase/RNAase free water for DNA content
quantification. Protein was quantified using the BioRad method (Thermo Scientific) and DNA was
quantified using the Hoechst (Hoechst 33258, Invitrogen-Molecular Probes) fluorescence assay
according to the manufacturer’s protocol. rASMC hypertrophy was determined as the ratio of
protein:DNA in each sample normalized to control samples.
FACS Analysis:
Flow cytometry was performed on a BD LSR II flow cytometer (BD Biosciences). rASMC
were grown to 80% confluence as indicated above and treated with 50 M H2O2 for 24 hr 37°C. Cells
were then trypsinized and resuspended in warm PBS (37°C) at a concentration of 1 x 106 cells/ml.
Forward scatter (FS) and side scatter (SS) were then measured recording 10,000 events per sample.
Quantification was then performed using FloJo. Density plots were gated to exclude debris and then
quadrant parameters were selected based on the size of 80 – 85 % of cells from control groups. The
percentage of events recorded in Quadrant 2 (FS +, SS +) were quantified for each group and taken as
a ratio of Quadrant 2 events percentage in control groups.
Thymidine Incorporation:
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Radiolabelled thymidine incorporation was performed as previously described
21
. Briefly,
rASMCs were plated on 24-well plates until 70 – 80% confluent and then serum starved as indicated
above. Cells were then incubated with 1 μCi/ml [3H]thymidine in the presence of PBS (vehicle) or 50
M H2O2 24 hr at 37°C, rinsed with PBS and exposed to 10% trichloroacetic acid (TCA) for 10 min.
TCA was removed and the cell monolayers were dissolved in 1N NaOH for the determination of
radioactivity.
Statistical Analyses:
Data are presented as means ± S.E.M. Data comparisons were performed with a Student’s ttest, one- or two-way-ANOVA including Bonferroni post-hoc analysis. Differences were deemed
statistically significant at a p < 0.05.
Supplemental Figure Legends:
Figure S1: Low (physiologic) concentration of H2O2 does not induce O2•– in rASMC. O2•–
production in pmol/min/mg protein quantified by SOD-inhibitable cytochrome c reduction in
membrane fractions of rASMC treated with 1 M H2O2. Data shown as means ± SEM, n = 4.
Figure S2: A spectrum of pharmacological inhibitors support NADPH oxidase as the main
source of H2O2-induced O2•– production in rASMC. Quantification of percent inhibition of the
maximal L-012 chemiluminescence in response to 50 M H2O2 in rASMC pre-incubated with the
indicated inhibitors and concentrations. Data are shown as means ± SEM, n = 3, * p < 0.05 vs H2O2
treated cells with no inhibitors.
Figure S3: Nox1 siRNA is specific for knockdown of Nox1 having no effect on Nox4 mRNA in
rASMC. Relative quantification normalized to 18S as assessed by qPCR of Nox1 (white bars) and
Nox4 (black bars) mRNA in rASMC transfected with 3 different siRNAs against Nox1 (Oligo1 – 3)
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or scrambled siRNA controls. Data are shown as means ± SEM, n = 4, * p < 0.05 vs scrambled
siRNA.
Figure S4: H2O2 does not affect expression of Nox1 or Aqp1 mRNA in rASMC. A)
Representative PCR for Nox1 and 18S mRNA of rASMC treated for the indicated times with 50 M
H2O2. Time 0 min indicates no treatment. B) Relative quantification normalized to 18S as assessed by
qPCR of Nox1 mRNA in rASMC treated with 50 M H2O2 for 1 hr. Data are shown as means ±
SEM, n = 3. C) Relative quantification normalized to 18S as assessed by qPCR of Aqp1 mRNA in
rASMC treated with 50 M H2O2 for 1 hr. Data are shown as means ± SEM, n = 3.
Figure S5: Aqp1 siRNA is effective for knockdown of Aqp1 mRNA in rASMC.
Relative
quantification as assessed by qPCR of Aqp1 mRNA in rASMC transfected with 3 different siRNAs
against Aqp1 (Oligo1 – 3) or scrambled siRNA controls normalized to 18S. Data are shown as means
± SEM, n = 3, * p < 0.05 vs scrambled siRNA.
Figure S6: Aqp1 siRNA transfection leads to a modest decrease in Nox1 protein in rASMC. A)
Representative Western blot for Nox1 and total actin of rASMC transfected with Scr. or Aqp1
siRNA. B) Quantification of fluorescence intensity of Nox1 bands obtained in A normalized to totalactin. C) Relative quantification normalized to 18S as assessed by qPCR of Nox1 mRNA in rASMC
transfected with Scr. or Aqp1 siRNA. Data shown as means ± SEM, n = 3, *p<0.05 vs. Scr. siRNA.
Figure S7: H2O2 induces Ask1 phosphorylation in rASMC overexpressing Ask1 in a Nox1dependent mechanism. A) Representative Western blot for phospho-Ask1 (threonine-845) and totalAsk1 of lysates of rASMC transfected with Scr. or Nox1 siRNA, transduced with Ad-Tg-Ask1
adenovirus and treated with 50 M H2O2. B) Quantification of fluorescence intensity of phosphoAsk1 bands obtained in A normalized to total-Ask1. Data shown as means ± SEM, n = 4, *p<0.05 vs.
vehicle Scr. siRNA; #p<0.05 vs. H2O2 Scr. siRNA.
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Figure S8: H2O2 induces increase in cell size of rASMC in a Nox1-dependent mechanism. A)
Representative images of rASMC transfected with Scr. or Nox1 siRNA and treated with 50 M H2O2
for 24 hr. B) Quantification of average cell areas as fold increase from vehicle Scr. siRNA. Data
shown as means ± SEM, n= 32 - 52 from 4 independent experiments, *p<0.05 vs. vehicle Scr.
siRNA; **p<0.05 vs. H2O2 Scr. siRNA.
Figure S9: In rASMC over-expressing Ask1, Aqp1 does not affect H2O2-induced Ask1
phosphorylation. A) Representative Western blot for phospho-Ask1 (threonine-845) and total-Ask1
(Right) and quantification of fluorescence intensity of phospho-Ask1 bands normalized to totalAsk1(Left) of lysates of rASMC transfected with Scr. or Aqp1 siRNA, transduced with Ad-Tg-Ask1
adenovirus and treated with 50 M H2O2 for 10 min. B) Representative Western blot (Right) and
quantification of band fluorescence intensity (Left) of lysates of rASMC transfected and transduced
as in A and treated with 50 M H2O2 for 1 hr. Data shown as means±SEM, n=3, *p<0.05 vs. Scr.
siRNA vehicle.
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