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INTRODUCTION |
Reperfusion of ischemic myocardium results in an abrupt
aggravation of cardiomyocyte injury, demonstrated experimentally by the
re-introduction of oxygen into hypoxic myocardium (1). This injury is
due in part to the generation of reactive oxygen species (2, 3), and
the injury is limited by antioxidants and free radical scavengers (4).
Potential sources of reactive oxygen species generation during
ischemia/reperfusion of vascular tissue include superoxide
(O
2) via neutrophil NADPH oxidase (5, 6) or from leakage of
electrons from the electron transport chain in the mitochondria (7).
The O
2 produced is converted to hydrogen peroxide
(H2O2) by superoxide dismutase, a weak free radical that reacts with O
2 to generate the highly active
hydroxyl radical (·OH). Anthracyclines, such as doxorubicin
induce cardiac injury in a similar manner, in that they are metabolized
to the corresponding semiquinone free radical by broadly distributed
enzymes, flavin reductases (7, 8).
In addition to causing tissue injury, free radicals induce genes that
are protective, such as hemoxygenase-1, which generates the
antioxidant, biliverdin (9). Cyclooxygenase is likewise cytoprotective
in several tissues (10) and plays a role in preventing apoptosis (11).
Moreover, cyclooxygenase activity is known to be sensitive to free
radicals (12). In this study, we explored the expression of
cyclooxygenase in response to free radical-induced injury of primary
cardiomyocytes using H2O2 and doxorubicin, both of which induce cardiac prostaglandin
(PG)1 formation (13).
Cyclooxygenase (COX), the rate-limiting enzyme in prostaglandin
synthesis, exists as two isoforms (14, 15). COX-1 is present in most
cells and is responsible for constitutive PG formation, whereas COX-2
is largely absent but is induced by cytokines, growth factors, and
hormones (16). A recent study has reported the expression of COX-2 in
ischemic human myocardium and in dilated cardiomyopathy, but not in
normal cardiomyocytes (17). How COX-2 is induced in the heart and its
function in these conditions is unknown. Cardiac fibrosis has been
reported in mice where the COX-2 isoform has been disrupted (18),
suggesting that COX-2 expression may be protective.
The mitogen-activated protein kinases (MAPKs) are serine/threonine
protein kinases and 4 subfamilies have been described. One of the
subfamilies, extracellular regulated kinases (ERKs)1/2, is activated by
a variety of growth factors, cytokines, and phorbol esters, and
regulates cellular proliferation and differentiation (19, 20). In
cardiac myocytes activation of ERK1/2 regulates gene expression and is
implicated in the development of cellular hypertrophy (21, 22).
Oxygen-derived free radicals induce activation of ERK1/2 and a second
MAP kinase, p38 in cardiac myocytes and other cells (23, 24).
H2O2-induced activation of ERK1/2 is mediated
through the Ras/Raf-1/Mek pathway (23-25). PD098059, a selective
inhibitor of ERK1/2 activation, aggravates
H2O2-induced apoptosis of cardiomyocytes (23).
The mechanism of the protective role played by ERK1/2 activation is
unknown. We investigated a possible link between ERK1/2 activation and
COX-2 expression in cardiomyocytes injured by oxygen-free radicals and
used a transcription factor decoy approach to decipher the role of the
promoter elements NF-
B and AP-1 in COX-2 induction.
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EXPERIMENTAL PROCEDURES |
Reagents--
Culture medium (Dulbecco's modified Eagle's
medium/Ham's F-12), pancreatin, newborn calf serum (NBS), LDH assay
kit LD-L10, phorbol 12-myristate 13-acetate (PMA), hydrogen peroxide
(H2O2), doxorubicin (DX), polyethyleneglycol
catalase (PEG-CAT), polyethyleneglycol superoxide dismutase
(PEG-superoxide dismutase), Immobilon-P polyvinylidene difluoride
membranes, 3,3'-diaminobenzidine tetrahydrochloride, Harris Hematoxylin
solution modified, permount and common laboratory chemicals were from
Sigma. Type II collagenase was from Worthington Biochemical Corp.,
Cambridge, MA. Deuterated eicosanoid standards, NS-398, fluprostenol
(PGF2
analog), and arachidonic acid were obtained from
Cayman Chemical Co., Ann Arbor, MI. Goat polyclonal anti-COX-1 antibody
was obtained from Oxford Biomedical Research Inc., MI. Mouse monoclonal
anti-COX-2 antibody (R6), which cross-reacts with the rat COX-2, was a
kind gift from Dr. Isakson, Monsanto, St. Louis, MO. Mouse monoclonals
anti-ERK1/2 were obtained from Transduction Laboratories, Lexington,
KY. Secondary anti-goat and anti-mouse biotinylated antibodies,
chemiluminescence reagents for assaying Western blot detection, and
p44/p42 MAP kinase assay kit were from New England Biolabs (UK) Ltd.,
Hertfordshire, UK. PCR prep kits were obtained from Promega,
Southhampton, UK. Primers for PCR and oligonucleotides for
transcription factor decoy experiments were purchased from Genosys
Biotech Inc., Cambridge, UK. Iloprost was from Schering AG, Berlin,
Germany. ERK1/2 inhibitor PD098059 and p38 MAP kinase inhibitor
SB203580 were obtained from Calbiochem, La Jolla, CA.
Cell Culture--
Primary cultures of neonatal rat
cardiomyocytes were prepared by a modification of the method originally
described by Simpson et al. (26). Briefly, the hearts from 1 to 3-day-old Wistar rats were minced and dissociated with approximately
80 units/ml type II collagenase and 0.06% pancreatin. Dispersed cells
were incubated in 25-cm2 flasks for 30 min at 37 °C in a
CO2 incubator. Nonattached viable cells were collected and
seeded into fresh flasks at a density of 1.5 × 105
cells/ml. To obtain a near-pure cardiomyocyte preparation, cells were
incubated in Dulbecco's modified Eagle's medium with Ham's F-12
supplemented with 1.2 g/liter sodium hydrogen carbonate, 10% (v/v) NBS
containing 1 mM pyruvic acid, 1 µg/ml transferrin, 10 µg/ml insulin, 10 ng/ml sodium selenite, 250 µM vitamin
C, 100 µM 5-bromo-2-deoxyuridine, 100 units/ml penicillin
G, and 100 µg/ml streptomycin. This medium was replaced with medium
containing no NBS at 48 h. The cells were incubated for 24 h
before addition of the test compounds.
Treatment of Cells with PMA--
On day 4 following the
isolation, cardiomyocytes grown in 25-cm2 flasks were
rinsed twice with PBS. PMA was added to medium without NBS and
incubated with cells for 6 h. For negative controls,
cardiomyocytes were incubated with medium alone. Following treatment
the medium was removed and cells were washed with PBS and medium
containing COX inhibitors were then added: 200 µM aspirin
for 30 min or 1 µM NS-398 for 45 min. All cells were then
washed twice with PBS and 2 ml of HBSS was added containing 50 µM arachidonic acid (peroxide free) for 10 min to measure
the maximum amount of prostaglandin formed. The supernatants were
stored at
70 °C for PG analysis.
Treatment of Cells with Hydrogen Peroxide and
Doxorubicin--
On day 4 following isolation, the cardiomyocytes were
rinsed twice with PBS. H2O2, 50 µM and 0.5 mM, or 100 µg/ml DX (172 µM) was added to medium without NBS and incubated with
cells for 10 or 80 min, respectively. For negative controls
cardiomyocytes were incubated with medium alone for equivalent amounts
of time. Oxygen-free radical scavengers PEG-CAT (200 units/ml) and
PEG-superoxide dismutase (200 units/ml) were added to cells 1 h
before treating with DX. The medium was then removed, the cells washed
with PBS and fresh medium added to allow cells recover for 10 min,
3 h, and 10 h. At these time points medium was removed and
stored at
70 °C for LDH analysis. The cells were then washed with
PBS and medium containing COX inhibitors were added: 200 µM aspirin for 30 min or 1 µM NS-398 for 45 min. All cells were then washed twice with PBS and 2 ml of HBSS was
added containing 50 µM arachidonic acid (peroxide-free)
for 10 min to measure the maximum amount of PG formed. This HBSS was
stored at
70 °C for analysis. The cells were then washed twice
with PBS and either lysed in protein 1% Triton X-100 lysis buffer and
stored at
20 °C for Western blot analysis or scraped into
Tri-Reagent and stored at
70 °C for RNA isolation. In further
experiments cells were treated with 1 µM NS-398 alone, or
10 µg/ml DX (17.2 µM) with or without 1 µM NS-398, or with 10 µg/ml DX in the presence of 200 units/ml PEG-CAT and 200 units/ml PEG-superoxide dismutase for 24 h. As above the medium was removed and stored at
70 °C for LDH
analysis, the cells washed with PBS and 2 ml of HBSS was added
containing 50 µM arachidonic acid for 10 min. The HBSS
was stored at
70 °C for PG analysis. The cells were washed and
scraped into Tri-Reagent and stored at
70 °C for mRNA analysis.
Treatment of Cells with Iloprost, or Fluprostenol, followed by
0.5 mM H2O2 or 100 µg/ml
Doxorubicin--
Cells were treated with 30 nM iloprost
alone, or with 1-100 nM iloprost, or medium alone
(positive control) for 24 h, before treating with 0.5 mM H2O2 or 100 µg/ml DX, and LDH
released into medium was measured. Cells were also treated both with
the PGF2
analog, 30 µM fluprostenol alone,
or 30 µM fluprostenol with 0.5 mM
H2O2 or 100 µg/ml DX, and LDH released into
medium was measured. The cells were subsequently scraped into
Tri-Reagent for COX-2 mRNA analysis.
Treatment of Cells with H2O2 or Doxorubicin and
the ERK Inhibitor PD098059 or the p38 MAP Kinase Inhibitor
SB203580--
Cells were pretreated with 50 µM PD098059
or 20 µM SB203580 for 1 h, before being exposed to
50 µM H2O2 or 100 µg/ml DX with PD098059 or SB203580 for 10 or 80 min, respectively. Following exposure
cells were allowed to recover in serum-free medium with 50 µM PD098059 or 20 µM SB203580 for 3 h.
As a positive control cardiomyocytes were incubated with 50 µM H2O2 or 100 µg/ml DX alone,
for 10 or 80 min, respectively, followed by a 3-h incubation of cells
in serum-free medium. As discussed above, all cells were washed with
PBS and 2 ml of HBSS was added containing 50 µM
arachidonic acid for 10 min. The HBSS was stored at
70 °C for PG
analysis. The cells were washed and either lysed in protein lysis
buffer or scraped into Tri-Reagent for mRNA analysis.
ERK1/2 Activation by H2O2 and
Doxorubicin--
Cells were treated with 50 µM
H2O2 or 100 µg/ml DX and 50 µM
PD098059 or 20 µM SB203580 with the same positive and
negative controls. All cells were then washed with PBS and ERK1/2
activity was measured using p44/p42 MAP kinase assay kit.
Transcription Factor Decoys and COX-2
Expression--
Transcription factor decoy oligomers were synthesized
by a method previously described by Schmedtje et al. (27)
in vitro by annealing the complementary strands in 1 × annealing buffer (20 µM Tris-HCl, pH 7.5, 20 µM MgCl2, and 50 µM NaCl). The
mixture was heated to 80 °C and allowed to cool to room temperature
slowly over 3 h. The oligomers were designed from two NF-
B
binding regions and one AP-1-like binding region on the rat COX-2
promoter. The NF-
B sequences used were, the distal site to the COX-2
promoter, (NF-
B-1)
413 5'-GAGAGGCAAGGGGATTCCCTTAGTTAG-3'
386 and
the proximal site to the COX-2 promoter, (NF-
B-2)
98
5'-GGGGGTGGGGAAAGCCGAGGCGGAAA-3'
72. The AP-1-like sequence was
173
5'-TCATTTGCGTGAGTAAAGCCTGCC-3'
149. A double-stranded DNA with a
scrambled sequence (5'-CAGGAGAGTATCCTGCGATGCATCTGCT-3') in the place of
the NF-
B and AP-1-like elements was used as a control in the decoy
experiments. Decoy naked double-stranded DNA was placed in the medium
at a concentration of 20 µM 1 h before exposing
cells to 50 µM H2O2 for 10 min,
or 100 µg/ml DX for 80 min. For the 3-h recovery fresh decoy naked
double-stranded DNA was added to medium at 20 µM,
enabling sequestration of the transcription factor in the cytoplasm. As
discussed under "Treatment of Cells with Hydrogen Peroxide and
Doxorubicin," all cells were washed with PBS and 2 ml of HBSS was
added containing 50 µM arachidonic acid for 10 min. The
HBSS was stored at
70 °C for PG analysis. The cells were washed
and either lysed in protein lysis buffer or scraped into Tri-Reagent
for mRNA analysis.
Reverse Transcription Polymerase Chain Reaction (PCR)--
This
procedure was used to compare the expression of COX-1 and COX-2
mRNAs between untreated and treated cells. Each pair of sense and
antisense primers were designed to span at least one intron of the gene
to exclude contaminating genomic DNA. Primers used were:
glyceraldehyde 3-phosphate dehydrogenase (GAPDH)
5'-AACCCATCACCATCTTCCAGGAGC-3' (sense) and
5'-CACAGTCTTCTGAGTGGCAGTGAT-3' (antisense); COX-1 5'-TCACAAGAGTACAGCTATGAGCAGT-3' (sense) and
5'-TGGGCTGGCACTTCTCCAGCATCAG-3' (antisense); COX-2
5'-CCCTGCTGGTGGAAAAGCCTCGTCC-3' (sense) and 5'-TACTGTAGGGTTAATGTCATCTAG-3' (antisense). Total RNA isolated from Tri-Reagent was reverse transcribed into cDNA and this was used as template for the PCR. RNA samples of 1 µg were denatured at
65 °C for 10 min. 100 ng of random hexamers, 1 × reverse
transcription buffer, 0.1 M dithiothreitol, 25 µM deoxyribonucleoside triphosphates, and 200 units of
Moloney murine leukemia virus reverse transcriptase were added and
incubated at 37 °C overnight in a reaction volume of 20 µl. The
reverse transcription reaction was stopped by heating to 95 °C for 5 min. A 1:10 volume of the generated cDNA reaction was used in the
subsequent amplification reaction. PCR was performed in a 50-µl
volume with 1.5 mM magnesium chloride, 1 mM
deoxynucleoside triphosphates, 0.5 µM of each sense and
antisense primer, and 1 unit of Taq polymerase. The reaction
cycles were denaturing at 94 °C for 1 min, annealing at 56 °C for
GAPDH and COX-2 for 1 min, annealing at 52 °C for COX-1 for 1 min,
and extension at 72 °C for 1 min. PCR cycle profiles for GAPDH,
COX-1, and COX-2 were performed. Products were run on agarose gels
(1.2%) with PGEM DNA markers. Polaroid instant photographs were taken
of the gels with a Polaroid DS34 direct screen instant camera.
Expression of COX-1 and COX-2 were normalized with respect to GAPDH
from parallel samples. In order to verify that the band produced at the
correct size was rat COX-2 cDNA, this band was excised from gel,
purified, cloned into PCR 2.1 vector, and sequenced (ABI Prism 310 Genetic Analyzer, Perkin-Elmer).
Western Blotting--
Lysates were prepared by treating cells
with lysis buffer. Protein concentration was measured using a protein
Bio-Rad assay, according to the manufacturer's procedure. Lysate
samples (20 µg of protein/lane) were applied to 10%
SDS-polyacrylamide minigels, electrophoresed, and transferred to
nitrocellulose membranes overnight at 40 V constant voltage at 4 °C
with use of a Western blot transfer apparatus. Immunostaining for COX
expression was performed with a goat polyclonal anti-COX-1 antibody and
a mouse monoclonal anti-COX-2 antibody, (R6), raised against the
recombinant full-length mouse (m) COX-2 expressed by baculovirus. R6
antibody is pre-absorbed with COX-2 (21 µM) but not COX-1
(25 µM) (28). Immunostaining for ERK1/2 was performed
with mouse monoclonal antibodies and developed with a horseradish
peroxidase Western blot detection kit.
LDH Assay--
Cytotoxicity was assessed by measuring LDH in the
culture medium spectrophotometrically. The LDH release was standardized with a cell injury index defined at (A-B)/(C-B) × 100, where A = LDH activity in the test sample; B = LDH activity measured in media with no cells (0% control), and C = LDH activity in samples from wells in which cells were lysed with Triton X-100 (100% control).
Determination of Eicosanoids by Gas Chromatography/Mass
Spectrometry (GC/MS)--
GC/MS was used to examine the range of PG
formed by resting and PMA-stimulated cardiomyocytes. For each sample,
200 µl of supernatant was removed and spiked with deuterated internal
standards. Following 15 min of equilibration, the pH was lowered to pH
3 with 10% formic acid and the samples were extracted with ethyl acetate and dried under vacuum. The methoxylamine derivative was formed
by adding 50 µl of a solution containing 0.5% methoxylamine in
pyridine and incubating overnight at room temperature. The sample was
then dried under vacuum. Derivitization to the pentafluorobenzyl ester
was accomplished by adding 10 µl of diidopropylethylamine and 20 µl
of 10% pentafluorobenzyl bromide in acetronitrile to the methoxylamine
derivative of the sample which was then allowed to stand overnight at
room temperature. The sample was again dried under vacuum before being
resuspended in 20 µl of
N'-O'-bis(trimethylsilyl)- acetamide and
incubated at 37 °C for 30 min. The samples were redried under vacuum
and brought up in 15 µl of dodecane. GC/MS was performed on a Varian
3400 gas chromatograph (GC) linked to a Finnigan Incos XL mass
spectrometer (MS) operated in the negative ion, chemical ionization
mode, using methane as the reagent gas. Source and interface
temperatures were 150 and 310 °C, respectively. The gas
chromatograph was equipped with a 30-m MS5 capillary column and
operated in splitless mode, with an injector temperature of 250 °C.
Following sample injection (3 µl), the GC temperature was ramped from
190 to 310 °C at 20 °C/min. Analytes were monitored by selected
monitoring of the mass ion, using INCOS data system.
Determination of Eicosanoids by Enzyme Immunoassay
Technique--
GC/MS demonstrated that 6-keto-PGF1
was
the main PG metabolite generated in neonatal cardiomyocytes. Thereafter
6-keto-PGF1
was measured by enzyme immunoassay (Assay
Design, Ann Arbor, MI).
Statistical Analysis--
All quantitative data are expressed as
mean ± S.E. The data were analyzed by parametric unpaired
Student's t test. p values < 0.05 were
considered statistically significant.
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RESULTS |
COX Isoform Expression and Product Formation in Rat Neonatal
Cardiomyocytes--
Morphological examination on the third day
following isolation showed a synchronous and rhythmically contracting
monolayer of cardiomyocytes. GC/MS analysis showed that the primary
prostaglandin product in cardiomyocytes is prostacyclin
PGE2 >PGF2
> TXB2 (Table
I). Similar levels of PG formation by
cardiomyocytes have been reported (29). Formation of both
6-keto-PGF1
and PGE2 was significantly
enhanced in response to PMA but neither PGF2
nor
TXB2 were altered (Table I).
Selective inhibition of COX-2 activity by 1 µM NS-398
specifically abolished the PMA-induced increase in
6-keto-PGF1
and PGE2, demonstrating that the increase in these PGs was COX-2-dependent. Inhibition of
COX activity by 200 µM aspirin prevented TXB2
generation, showing that its generation was
COX-1-dependent. PGF2
appears to be
generated non-enzymatically.
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Table I
Prostaglandin generation (pg/mg of protein/10 min) in neonatal rat
cardiomyocytes
The main prostaglandins generated by cardiomyocytes were
6-keto-PGF1 and PGE2, which were PMA-induced. This
induction was blocked by NS-398, a specific COX-2 inhibitor.
PGF2 or TXB2 formation was not significantly
enhanced by PMA stimulation. Cells that were PMA-treated with aspirin
show that PGF2 is nonenzymatically generated and
TXB2 production was mostly COX-1-dependent.
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H2O2-induced COX-2 Expression at 3 h--
Cardiomyocytes were exposed to 50 µM
H2O2 for 10 min which did not effect cell
viability. RT-PCR analysis (Fig.
1A) showed that
H2O2-induced COX-2 expression at 3 h did
not affect mRNA levels of COX-1 or the housekeeping gene, GAPDH.
There was a corresponding increase in COX-2 protein, whereas the
expression of COX-1 did not change (Fig. 1B).
H2O2 also induced 6-keto-PGF1
production at 3 h, with levels returning to baseline by 10 h
(Fig. 1C), as did the expression of COX-2 protein and
mRNA. 1 µM NS-398 had no effect on product formation
at baseline or at 10 min of recovery, but abolished the increase in
6-keto-PGF1
at 3 h, demonstrating that this
increase was COX-2-dependent. NS-398 did not effect the
expression of COX-2 message and was therefore only inhibiting product
activity (data not shown). Aspirin, a non-selective COX inhibitor,
reduced the amount of 6-keto-PGF1
formed at all time
points.

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Fig. 1.
COX-2 expression in isolated primary rat
neonatal cardiomyocytes treated with 50 µM H2O2 for 10 min (n = 3). Before treatment with
H2O2 (lane 1), and 10 min
(lane 2), 3 h (lane 3), and 10 h
(lane 4) following washout of
H2O2. A, mRNA transcript levels
of COX-1 and COX-2 determined by RT-PCR analysis. GAPDH was used as an
internal control. B, Western blot analysis for COX-1 and
COX-2. COX-2 was induced at 3 h and returned to baseline by
10 h. C, 6-keto-PGF1 generation in
response to H2O2 in the presence and absence of
inhibitors, 200 µM aspirin and 1 µM NS-398.
The data are expressed in picograms/milligrams of protein generated
over 10 min following the addition of 50 µM arachidonic
acid. H2O2-induced 6-keto-PGF1
generation at 3 h ( , p < 0.05 versus
controls). This increase was reduced by the selective COX-2 inhibitor,
NS-398 (1 µM) and aspirin (200 µM) (*,
p < 0.05; **, p < 0.005 versus H2O2 treated).
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Doxorubicin Induces COX-2 Expression Due to Free Radical
Formation--
Addition of 10 or 100 µg/ml doxorubicin to cells
resulted in a marked and rapid reduction in their rate of contraction.
Once doxorubicin was removed, however, and cells were allowed to
recover for 20 min, a normal rate of contraction was restored. Addition of 100 µg/ml doxorubicin to cells for 80 min, followed by a 3-h recovery, did not cause cells to apoptose appreciably. However, 10 µg/ml doxorubicin over 24 h induced cell shrinkage, nuclear condensation, formation of apoptotic bodies, and blebs in the cell
membrane in about 10% of cells. 100 µg/ml doxorubicin induced a
time-dependent change in COX-2 mRNA levels (Fig.
2A), detectable after 10 min
and peaking after 3 h recovery, without any change in COX-1 or
GAPDH mRNA levels. 200 units/ml PEG-superoxide dismutase and
PEG-CAT prevented COX-2 expression, demonstrating a role for free
radicals in doxorubicin-mediated COX-2 induction. Doxorubicin (100 µg/ml) also caused an increase in 6-keto-PGF1
formation after 3 h (Fig. 2B). This increase in
6-keto-PGF1
was abolished by 1 µM NS-398
without preventing the expression of COX-2, demonstrating that the
increase in product formation was COX-2-dependent. The increase in 6-keto-PGF1
was also abolished by
pretreatment with 200 units/ml PEG-superoxide dismutase and
PEG-CAT.

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Fig. 2.
Induction of COX-2,
6-keto-PGF1 generation, and cell
injury by doxorubicin. Cells were exposed to 100 µg/ml
doxorubicin for 80 min, washed, and allowed to recover for the times
shown, (n = 3). A, RT-PCR for COX-1, COX-2,
and GADPH. There was no induction of COX-1 but COX-2 was induced at 10 min and at 3 h. By 10 h COX-2 expression had returned to
normal. B, the generation of 6-keto-PGF1 was
induced at 3 h after doxorubicin exposure ( , p < 0.05 versus controls). This increase was abolished by the
COX-2 inhibitor NS-398 (1 µM) and by free radical
scavengers, PEG-superoxide dismutase (SOD) (200 units/ml)
and PEG-CAT (200 units/ml). The non-selective COX inhibitor aspirin
inhibited 6-keto-PGF1 formation at all time points (**,
p < 0.005 versus doxorubicin-treated).
C, cell injury in response to doxorubicin was measured as
release of LDH and increased at both 3 and 10 h ( ,
p < 0.05 versus controls). Pretreatment
with PEG-superoxide dismutase and PEG-CAT prevented the increase in LDH
(*, p < 0.05 versus doxorubicin).
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The release of LDH into cell medium was measured as a marker of cell
injury (Fig. 2C). Following the initial 80-min exposure of
cells to 100 µg/ml doxorubicin, there was no detectable rise in LDH
at 10 min. After 3 h, there was a significant increase indicating
that cell injury had occurred. There was no further increase in LDH
released from 3 to 10 h. The increase in LDH release was
suppressed by pretreatment with PEG-superoxide dismutase and PEG-CAT.
PEG-superoxide dismutase alone increased the amount of LDH released,
demonstrating the role of the hydroxyl radical in doxorubicin-mediated
cell injury.
The lower concentration of 10 µg/ml doxorubicin also induced COX-2,
but at a later time point. Doxorubicin (10 µg/ml) induced COX-2
message at 24 h without affecting mRNA levels of COX-1 or GAPDH. Doxorubicin (10 µg/ml) also caused an increase in
COX-2-dependent 6-keto-PGF1
formation (Fig.
3, A and B). Again
PEG-superoxide dismutase and PEG-CAT prevented COX-2 induction and
product formation, suggesting a free radical-dependent
mechanism. At this low drug concentration a continual accumulation of
free radical metabolites of doxorubicin may be responsible for the
COX-2 induction. It is also possible, however, that doxorubicin at this
low concentration causes a steady increase in ceramide production by
the cardiomyocyte (26) which is reported to induce apoptosis and
mitochondrial H2O2 production (27).

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Fig. 3.
Cardiomyocyte response to 10 µg/ml doxorubicin for 24 h. A,
RT-PCR for COX-1, COX-2, and GADPH. 10 µg/ml DX over 24 h
induced COX-2 transcript levels which was prevented by PEG-superoxide
dismutase (SOD) and PEG-CAT. B, doxorubicin also
induced 6-keto-PGF1 formation at 24 h ( ,
p < 0.05 versus control) that was abolished
by the selective COX-2 inhibitor NS-398, aspirin, and PEG-superoxide
dismutase and PEG-CAT (*p < 0.05, p < 0.005). C, cell injury in response to doxorubicin was
measured as release of LDH. There was a significant rise, ( ,
p < 0.05 versus control) in the amount of
LDH released after 24 h, which was prevented by treatment with
PEG-superoxide dismutase and PEG-CAT (**, p < 0.005).
Treatment of cells with the selective COX-2 inhibitor NS-398 alone over
the 24-h period did not affect LDH release, however, NS-398 enhanced
significantly (*, p < 0.05) the release of LDH by
doxorubicin.
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10 µg/ml doxorubicin also caused the release of LDH which was
prevented by PEG-superoxide dismutase and PEG-CAT (Fig. 3C). The COX-2 inhibitor NS-398 (1 µM) alone did not increase
the release of LDH above that of controls. The release of LDH, however,
was aggravated by 1 µM NS-398 (Fig. 3C),
suggesting that a COX-2-dependent product protects the
cells from injury.
Iloprost Protects Cardiomyocytes from Free Radical-induced
Damage--
Since COX-2-dependent PGs protect
cardiomyocytes from injury, we examined the effects of iloprost (a
stable analog of prostacyclin) on LDH release by cells exposed to high
concentrations of H2O2 (as 50 µM
H2O2 did not cause a substantial increase in
LDH release following treatment). 30 nM iloprost reduced
the amount of LDH released by H2O2 and
doxorubicin-induced cell damage (Fig. 4, A and B). In contrast, an analog of
PGF2
, 30 µM fluprostenol, did not prevent
LDH release from cardiomyocytes, demonstrating that the reduction in
LDH release was specific for iloprost.

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Fig. 4.
LDH release on pretreating cardiomyocytes
with iloprost or fluprostenol followed by exposure to 0.5 mM H2O2 or 100 µg/ml doxorubicin (n = 3).
A, 0.5 mM H2O2 for 10 min caused a significant increase ( , p < 0.005 versus control) in the amount of LDH released. Pretreating
the cells with increasing concentrations of iloprost prevented LDH
release, however, the PGF2 analogues, 30 µM fluprostenol, did not prevent LDH release.
B, doxorubicin caused a significant increase ( ,
p < 0.005 versus control) in the amount of
LDH released. Again pretreating the cells with increasing
concentrations of iloprost prevented LDH release (*, p < 0.05, p < 0.005), while 30 µM
fluprostenol had no effect.
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Free Radicals Cause ERK Activation, Which Mediates COX-2
Expression--
Incubation of cardiomyocytes with 50 µM
H2O2 or 100 µg/ml doxorubicin increased
ERK1/2 activity (Fig. 5A). The
increase in ERK1/2 activity was suppressed in the presence of the
ERK1/2 inhibitor PD098059 (50 µM) which also prevented
the induction of COX-2 protein, mRNA, and product in response to
H2O2 or doxorubicin (Fig.
6, B, C, and
D). In contrast, the p38 MAP kinase inhibitor, SB203580 (20 µM), did not prevent COX-2 induction (Fig. 5,
B, C, and D), suggesting that COX-2
expression was specifically ERK1/2 mediated.

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Fig. 5.
Cardiomyocyte response to inhibition of
ERK1/2 activity and inhibition of p38 MAP kinase activity 3 h
following treatment with 50 µM
H2O2 for 10 min or 100 µg/ml doxorubicin for 80 min
(n = 3). Untreated cells (lane
1), 50 µM H2O2 (lane
2), 100 µg/ml doxorubicin (lane 3), 50 µM H2O2 plus 50 µM
PD098059 (lane 4), 100 µM doxorubicin plus 50 µM PD098059 (lane 5), 50 µM
H2O2 plus 20 µM SB203580
(lane 6), 100 µM doxorubicin plus 20 µM SB203580 (lane 7). A, Western blot analysis
of ERK1/2 protein and activity shows cardiomyocytes express p44/p42
ERKs protein (1). ERK1/2 activity increased in response to
H2O2 (2) or doxorubicin
(3) after 3 h (n = 3). The ERK1/2
specific inhibitor PD098059 (50 µM )prevented ERK1/2
activation in response to H2O2 (4)
and doxorubicin (5). B, RT-PCR for COX-1 and
COX-2. C, Western blot for COX-1 and COX-2 protein. The
ERK1/2 specific inhibitor PD098059 (50 µM) prevented
COX-2 expression in response to H2O2
(4) or doxorubicin (5), whereas the p38 MAP
kinase specific inhibitor, SB203580 (20 µM), had no
effect (6, 7). Inhibition of ERK1/2 or 38 MAP kinase did not effect
COX-1 expression. D, 6-keto-PGF1 formation by
cardiomyocytes. PG formation was induced by
H2O2 and doxorubicin (2, 3) ( ,
p < 0.05 versus control). Pretreatment with
the ERK1/2 inhibitor PD098059 abolished this increase (*,
p < 0.05 versus corresponding
H2O2 or doxorubicin-treated cells)
(4 and 5), whereas the p38 MAP kinase inhibitor
SB203580 had no effect (6 and 7).
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Fig. 6.
Effect of transcription factor
NF- B and AP-1 decoys on
H2O2 and doxorubicin induced COX-2 expression
(n = 3). Non-treated cells (lane 1),
50 µM H2O2-treated (lane
2), 100 µg/ml doxorubicin-treated (lane 3),
H2O2-treated + scrambled oligonucleotide
(lane 4), doxorubicin-treated + scrambled
oligonucleotide (lane 5),
H2O2-treated + AP-1 oligonucleotide (lane
6), doxorubicin-treated + AP-1
oligonucleotide (lane 7),
H2O2-treated + NF- B-1 oligonucleotide
(lane 8), doxorubicin-treated + NF- B-1
oligonucleotide (lane 9),
H2O2-treated + NF- B-2 oligonucleotide
(lane 10), doxorubicin-treated + NF- B-2
oligonucleotide (lane 11). A, RT-PCR; and B,
Western blot analysis of COX-1 and COX-2 expression.
H2O2 and doxorubicin induced expression of
COX-2 without altering COX-1 or GADPH (lanes 2 and
3). The scrambled decoy oligomers did not prevent COX-2
expression (lanes 4 and 5). However, the AP-1
double-stranded decoy oligomer prevented COX-2 expression in response
to either H2O2 or doxorubicin (lanes
6 and 7). A double-stranded decoy oligomer
corresponding to the distal NF- B-1 had no effect on COX-2 expression
(lanes 8 and 9). A double-stranded decoy oligomer
corresponding to the proximal NF- B-2 reduced but did not abolish
COX-2 induction by H2O2 or doxorubicin
(lanes 10 and 11). C, corresponding changes in
6-keto-PGF1 formation. H2O2 or
doxorubicin induced 6-keto-PGF1 formation (**,
p < 0.005 versus controls). The increase
was abolished by the AP-1 double-stranded decoy oligomer and reduced by
the proximal NF- B-2 double-stranded decoy oligomer (*,
p < 0.05).
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The Transcription Factors AP-1 and NF-
B Are Responsible
for ERK Activation and Expression of COX-2 in
Cardiomyocytes--
Cardiomyocytes were treated with double-stranded
DNA oligonucleotides (all at 20 µM) corresponding to
COX-2 promoter elements to act as decoys for transcription factors that
may be generated and so localize the elements responsible for the
induction of COX-2. A decoy corresponding to the AP-1-like promoter
element prevented COX-2 expression in response to
H2O2 or doxorubicin (Fig. 6, A-D).
An oligonucleotide decoy corresponding to the proximal NF-
B-2 also
reduced, but did not abolish the induction of COX-2, whereas an
oligonucleotide decoy corresponding to the distal NF-
B-1 COX-2
promoter had no effect.
 |
DISCUSSION |
Cyclooxygenase-2 has been identified recently in human
cardiomyocytes in areas of myocardial infarction and from individuals with dilated cardiomyopathy, whereas no COX-2 was found in normal hearts (17). The mechanism for the induction of COX-2 in cardiomyocytes is unknown. In addition to growth factors and cytokines, shear stress,
hypoxia, calcium ionophore, bradykinin, thrombin, and angiotensin II
(30-35) induce COX-2 expression. Oxidant stress may also induce COX-2
(12). Feng et al. (12) showed that the inhibition of
prostaglandin synthesis in rat mesangial cells by free radical
scavengers was due to the suppression of COX-2 expression.
As in the human cardiomyocyte, COX-1 was the only isoform expressed in
resting rat cardiomyocytes while COX-2 was induced by treatment with a
phorbol ester. COX-2 was also induced by two oxidants, doxorubicin at a
therapeutically relevant concentration, and brief exposure to a
relatively low concentration of H2O2. Doxorubicin is an anthracycline used as a chemotherapeutic agent and is
well recognized to induce a dilated cardiomyopathy (36). A hydroxyl
radical originating from a superoxide radical has been implicated in
anthracycline-mediated cardiotoxicity (7, 8). Alternatively, toxicity
may be due to accumulation of ceramide, a lipid formed when cells are
exposed to anthracyclines and which may induce mitochondrial
H2O2 production (37). Doxorubicin also induced
toxicity in cultured cardiomyocytes detected as release of LDH. Both
the expression of COX-2 and the release of LDH were abolished by the
combination of superoxide dismutase and catalase confirming a free
radical-dependent mechanism.
Oxygen-free radicals have indiscriminate effects on a variety of
cellular targets including lipids, proteins, and oligonucleotides, resulting in cell death. At lower concentrations, however, oxygen-free radicals activate very specific pathways that in turn regulate the
expression of genes influencing cell survival. Exogenous free radicals
have been shown to activate p38 MAP kinase in several cell systems and
less often to activate ERK1/2. Xia et al. (38) reported that
free radical-induced activation of ERKs promotes cell survival, whereas
activation of JNK and p38 MAPK induce apoptosis. Similarly,
ceramide-induced activation of JNK is important for the induction of
apoptosis (39). These results suggest that ERKs have a protective role,
while activation of p38 MAPK/JNK leads to induction of apoptotic death
on exposure to free radicals. The pattern of MAP kinase activation in
response to oxidant stress varies between cells even in the same
species. In rat vascular smooth muscle cells free radicals activate p38
MAP kinase but not ERK1/2 (40). In contrast, in rat neonatal
cardiomyocytes H2O2 specifically activates ERKs
through activation of the Src family of tyrosine kinases, Ras and RAF-1
(23). The activation of ERKs in these cells prevented
H2O2-induced apoptosis although the mechanism
was not identified. Similarly in our studies ERK1/2 was activated by
H2O2 and doxorubicin and was responsible for the activation of COX-2. Thus, PD098059, which prevents
MEK-dependent activation of ERK1/2, abolished the
expression of COX-2 mRNA and protein and the increase in
prostaglandin formation. In contrast, specific inhibition of p38 MAP
kinase had no effect.
The Src family of tyrosine kinases has been implicated in the induction
of COX-2 in response to cytokines and growth factors, but not
specifically the ERK kinase (41). The ERK1/2 kinase pathway, however,
has been identified as the common signaling pathway of serotonin or
phorbol ester-induced COX-2 expression in rat mesangial cells (42) and
both ERK kinase and MAPK p38 have been shown to play a role in
lysophosphatidic acid-mediated COX-2 expression by these cells (43). To
identify how ERK1/2 induced COX-2 in cardiomyocytes we examined three
putative sites on the promoter of the rat COX-2 gene. Two of the sites
are homologous to the NF-
B element, which is also found in the
promoters of human inflammatory genes, including the human COX-2 gene.
The third, the AP-1-like site is similar to a promoter element common to early response genes. In high concentration,
H2O2 induces translocation of NF-
B to the
nucleus and subsequent gene expression in an endothelial cell line
(27). This is by no means a ubiquitous event and in some cells there is
gene induction in response to H2O2 without NF-
B translocation (27). Here, we show that AP-1 is the major promoter element involved, as a decoy promoter AP-1 sequence abolished COX-2 induction. ERK1/2 may induce c-Fos expression which complexes in
a heterodimer with c-Jun forming the transcription factor that acts
through AP-1. This is thought to explain interleukin-1
induction of
nitric oxide in rat pancreatic islet cells, which is also ERK1/2 dependent (44). Moreover, increased activation of ERKs and of the AP-1
promoter has been shown in proliferating smooth muscle cells (45, 46).
A double-stranded oligonucleotide decoy identical to the proximal
NF-
B site showed that this site was also partially active, whereas
the distal NF-
B site was inactive. These findings implicate both the
proximal NF-
B and the AP-1 site, but suggest that the latter is the
critical promoter element. It is worth noting that both AP-1 and
NF-
B are activated by H2O2 in neonatal cardiomyocytes (47) and that intracellular oxidants may be the ultimate
messengers common to all signals that activate AP-1 and NF-
B
(48).
H2O2 and DX induced the release of LDH from
cardiomyocytes, and the selective COX-2 inhibitor NS-398 potentiated
this. In contrast, treating the cells with NS-398 alone did not affect the basal levels of LDH release. The injury response was prevented by
iloprost, a stable analogue of the principle cyclooxygenase product of
these cells, prostacyclin. The response to iloprost appeared to be
specific as it was dose dependent and no protection was seen with
fluprostenol, a stable analogue of PGF2
, which induces
hypertrophy of rat cardiomyocytes (49). These data suggest that
COX-2-dependent prostaglandins protect the cardiomyocyte from oxidant injury. Prostaglandins are cytoprotective in several tissues, including stomach (50, 51) and heart (52). COX-2 expression
also protects against apoptosis in a number of cancer cell lines (53)
and against nitric oxide-mediated apoptosis (54). Similarly, rats
injected with lipopolysaccharide (a known inducer of COX-2) are
protected from ischemia/reperfusion injury (55). Prostaglandins,
particularly metabolites of PGJ2, interact with the
peroxisomal proliferator activator receptor-
, a nuclear envelope
receptor that acts as a transcription factor for apoptosis regulating
genes (56). Prostacyclin has not been shown, however, to react with
this receptor, so the mechanism for COX-2 dependent protection of the
cardiomyocyte seen in our experiments is unknown.
Adaptive responses of bacteria to the potential toxic effects of
partially reduced oxygen metabolites (H2O2,
·OH, and O
2) include the transcriptional regulation of
oxidative stress genes (57). Similar mechanisms exist in eukaryocytic cells where reactive oxygen species cause expression of several enzymes
involved in oxidant metabolism such as the human glutathione peroxidase
gene (58, 59). The induction of COX-2 in cardiomyocytes may represent
an additional example of an adaptive response that protects the cell
from oxidant stress.