From the National Heart and Lung Institute Division,
Imperial College School of Medicine, Royal Brompton Campus, London SW3
6LY, United Kingdom and the § Division of Biomedical
Sciences, Imperial College School of Medicine, Charing Cross Campus,
London W6 8RF, United Kingdom
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ABSTRACT |
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"Stress-regulated" mitogen-activated protein
kinases (SR-MAPKs) comprise the stress-activated protein kinases
(SAPKs)/c-Jun N-terminal kinases (JNKs) and the p38-MAPKs. In the
perfused heart, ischemia/reperfusion activates SR-MAPKs. Although the
agent(s) directly responsible is unclear, reactive oxygen species are
generated during ischemia/reperfusion. We have assessed the ability of
oxidative stress (as exemplified by H2O2)
to activate SR-MAPKs in the perfused heart and compared it with the
effect of ischemia/reperfusion. H2O2 activated
both SAPKs/JNKs and p38-MAPK. Maximal activation by
H2O2 in both cases was observed at 0.5 mM. Whereas activation of p38-MAPK by
H2O2 was comparable to that of ischemia and
ischemia/reperfusion, activation of the SAPKs/JNKs was less than that
of ischemia/reperfusion. As with ischemia/reperfusion, there was
minimal activation of the ERK MAPK subfamily by
H2O2. MAPK-activated protein kinase 2 (MAPKAPK2), a downstream substrate of p38-MAPKs, was activated by
H2O2 to a similar extent as with ischemia or
ischemia/reperfusion. In all instances, activation of MAPKAPK2 in
perfused hearts was inhibited by SB203580, an inhibitor of p38-MAPKs.
Perfusion of hearts at high aortic pressure (20 kilopascals) also
activated the SR-MAPKs and MAPKAPK2. Free radical trapping agents
(dimethyl sulfoxide and N-t-butyl--phenyl nitrone)
inhibited the activation of SR-MAPKs and MAPKAPK2 by
ischemia/reperfusion. These data are consistent with a role for
reactive oxygen species in the activation of SR-MAPKs during
ischemia/reperfusion.
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INTRODUCTION |
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Ischemic and hypertensive myocardial disease is currently a major cause of mortality and morbidity. In these conditions, the heart is exposed to numerous cell stresses including increased production of reactive oxygen species (ROS),1 ionic imbalances, osmotic stress, mechanical stress, and metabolic deprivation (reviewed in Refs. 1-4). In numerous cell lines, primary cultures, and tissues, cellular stresses activate "stress-regulated" mitogen-activated protein kinases (SR-MAPKs) (reviewed in Refs. 5 and 6). There are two relatively well characterized SR-MAPK families, the stress-activated protein kinases (SAPKs) and the p38-MAPKs. SAPKs are alternatively known as the c-Jun N-terminal kinases (JNKs), although, strictly speaking, the JNK terminology applies to the human enzymes, whereas the SAPK terminology applies to the rat enzymes. Substrates for SAPKs/JNKs include the transcription factors c-Jun (7, 8), ATF-2 (9-12), and Elk-1 (13, 14). Phosphorylation of these transcription factors in their trans-activation domains leads to an increase in their ability to trans-activate transcription. p38-MAPK (15, 16) (alternatively known as cytokine-suppressive antiinflammatory drug-binding protein (17), reactivating kinase (18), Mxi2 (19), or stress-activated protein kinase-2 (20)) is a mammalian homolog of the yeast osmosensing protein kinase HOG-1. Like SAPKs/JNKs, p38-MAPKs also phosphorylate transcription factors (ATF2 (11), CHOP/GADD153 (21), and MEF2C (22)), increasing their trans-activating activity or altering their specificity. In addition, p38-MAPK phosphorylates and activates MAPK-activated protein kinases (MAPKAPKs) 2 and 3 (18, 23), which in turn phosphorylate the small heat shock proteins (Hsp25/27) (18, 23-25). This may modulate the cytoprotective activity of Hsp25/27 (26). The third subfamily of MAPKs (the extracellularly responsive kinases, ERKs) is involved in the regulation of cell growth and differentiation (reviewed in Refs. 27 and 28). In the heart (as in other tissues), these MAPKs are more strongly activated by growth-promoting stimuli (phorbol esters, G protein-coupled receptor agonists, and peptide growth factors) than by cell stresses, with the reverse situation applying to the SR-MAPKs (29-31).
We and others have recently shown that p38-MAPK and MAPKAPK2 are strongly activated by ischemia in the perfused rat heart (32, 33). On reperfusion, activation of these kinases is maintained, and in addition SAPKs/JNKs are activated (32-34). Consistent with this, SAPKs/JNKs are activated in neonatal cardiac myocytes subjected to hypoxia/reoxygenation but not hypoxia alone (35). The roles of these SR-MAPKs remain obscure. We are interested in signals that may potentially activate the SR-MAPKs in ischemic/reperfusion stress. During ischemia and on reperfusion of the ischemic myocardium, there is release of ROS as well as other factors (reviewed in Refs. 1, 2, and 4). Here, we have examined the potential of oxidative stress (as exemplified by perfusion with H2O2) to activate SR-MAPKs and MAPKAPK2 in the isolated rat heart and have compared the effects of H2O2 with those of ischemia and ischemia/reperfusion. Using antioxidants, we investigated the role of ROS in the activation of SR-MAPKs in ischemia/reperfusion. In addition, we have examined the effects of another pathophysiologically important stress (hypertensive stress) on activation of the SR-MAPKs.
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EXPERIMENTAL PROCEDURES |
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Materials--
Prestained molecular mass markers, biotinylated
anti-rabbit IgG, ECL blotting reagents, and Hyperfilm MP were from
Amersham International. [-32P]ATP was from NEN Life
Science Products. Protein A-Sepharose, N-t-butyl-
-phenyl nitrone (BPN), and other
biochemicals were from Sigma. SDS-polyacrylamide gel electrophoresis
reagents and Bradford (36) protein assay reagent were from Bio-Rad.
SB203580 was a gift from Dr. John C. Lee (SmithKline Beecham, King of
Prussia, PA). Nitrocellulose was from Schleicher & Schuell.
cAMP-dependent protein kinase inhibitory peptide (PKI,
amino acid sequence TTYADFIASGRTGRRNAIHD) was from Bachem. The MAPKAPK2
substrate peptide (KKLNRTLSVA (18, 37)) was synthesized by Severn
Biotech. General laboratory chemicals were from Merck. Antibodies to
JNK1, JNK2, and p38-MAPK were from Santa Cruz Biotechnology Inc. The
JNK1 antibody (sc-474) was a rabbit antibody raised to a C-terminal
region of human JNK1 (amino acids 368-384). Antibodies to JNK2 (sc-572
and sc-827) were raised to the full-length 424-residue protein and
amino acids 5-22 from human JNK2, respectively. The p38-MAPK antibody
(sc-535) was a rabbit antibody raised to amino acids 341-360 at the C
terminus of the mouse sequence. The N-terminal
trans-activation domain of human c-Jun (amino acid residues
1-135) and the catalytic domain of murine MAPKAPK2 (amino acid
residues 46-400) were expressed as glutathione
S-transferase (GST) fusion proteins in Escherichia coli and were purified by glutathione-Sepharose (Pharmacia)
chromatography (38). They were used without cleavage from the GST
moiety. The c-Jun(1-135) region encompasses the following residues
that can be phosphorylated by SAPKs/JNKs: Ser-63, Ser-73, Thr-91, and
Thr-93 (39). The MAPKAPK2(46-400) region encompasses the following residues that can be phosphorylated by p38-MAPK: Thr-222, Thr-272, and
Thr-334 (40).
Heart Perfusions-- Adult male (250-300 g) Sprague-Dawley rat hearts were perfused retrogradely at a pressure of 10 kilopascals (70 mm Hg) with Krebs-Henseleit bicarbonate-buffered saline (25 mM NaHCO3, 119 mM NaCl, 4.7 mM KCl, 2.5 mM CaCl2, 1.2 mM KH2PO4 (pH 7.6)) at 37 °C supplemented with 10 mM glucose and equilibrated with 95% O2/5% CO2. The temperature of the perfusates and hearts was maintained at 37 °C by the use of a water-jacketed apparatus. Coronary flows were determined at 10 min after cannulation and were also measured during and at the end of the experiments. All other times given refer to times following the appropriate equilibration period.
For experiments in which H2O2 was the only agent added, hearts were perfused for a 15-min equilibration period. H2O2 was added to the requisite concentration, and hearts were perfused for a further 5-30 min. For hearts subjected solely to simple global ischemia or to ischemia/reperfusion, the equilibration period was 15 min. The perfusion was then interrupted for 20 min by clamping the aortic perfusion line. Hearts ceased beating within 1 min of ischemia. Where indicated, ischemic hearts were reperfused for 10 min by reopening the aortic perfusion line. Hearts resumed beating within 1 min of reperfusion, and coronary flow returned to within 80% of control values (control, 13 ml/min/heart; reperfused, 11 ml/min/heart). Control hearts were perfused for up to 30 min after the pre-equilibration period without interruption to the perfusate flow. For hearts subjected to increased aortic pressure, the perfusion pressure was raised to 20 kilopascals (140 mm Hg) after the 15-min equilibration period, and the perfusions were continued for 20 min. When Me2SO, SB203580 (10 mM stock in Me2SO), or BPN (2.5 M stock in Me2SO) was used, the hearts were perfused for 15 min. Me2SO, SB203580/Me2SO, or BPN/Me2SO was then added, and the perfusions were continued for another 15 min before the addition of H2O2 or the imposition of ischemia. At the end of all perfusions, hearts were "freeze-clamped" between aluminum tongs cooled in liquid N2 and pulverized under liquid N2, and the powders were stored atImmunoprecipitation--
Heart powders were homogenized with 3 volumes of immunoprecipitation buffer (10 mM Tris/HCl (pH
7.4), 5 mM EDTA, 50 mM NaF, 50 mM
NaCl, 2 mM Na3VO4, 0.1% (w/v)
fatty acid free bovine serum albumin, 20 µg/ml aprotinin) containing
20 mM n-octyl--D-glucopyranoside and 1% (v/v) Triton X-100. The samples were extracted on ice (10 min)
and centrifuged (10,000 × g, 5 min, 4 °C). The
supernatants (150 µl) were incubated with 4 µl (0.4 µg) JNK1
antibody or 8 µl (0.8 µg) p38-MAPK antibody on a rotating wheel (2 h, 4 °C). Protein A-Sepharose was added (20 µl of a 50% slurry in
immunoprecipitation buffer), and the samples were rotated for a further
1 h. The samples were centrifuged (10,000 × g, 1 min, 4 °C), and the supernatants were removed and boiled with 0.33 volume of SDS sample buffer (0.33 M Tris-HCl (pH 6.8), 10%
(w/v) SDS, 13% (v/v) glycerol, 133 mM dithiothreitol, 0.2 mg/ml bromphenol blue). The pellet was washed in immunoprecipitation
buffer (3 × 150 µl, 4 °C), resuspended in 150 µl, and
boiled with 50 µl of SDS sample buffer. Samples (30 µl) were
assayed for JNK1 or p38-MAPK activity by the in gel kinase method.
In Gel Kinase Assays--
MAPKs in immunoprecipitates prepared
from heart extracts (see previous section) or in whole heart extracts
were assayed by the in gel kinase method (41). For analysis of whole
heart extracts, heart powders were homogenized with 3 volumes of buffer
A (20 mM -glycerophosphate (pH 7.5), 50 mM
NaF, 2 mM EDTA, 0.2 mM
Na3VO4, 10 mM benzamidine, 200 µM leupeptin, 10 µM trans-epoxy
succinyl-L-leucylamido-(4-guanidino)butane, 5 mM dithiothreitol, 300 µM
phenylmethylsulfonyl fluoride, 1% (v/v) Triton X-100) and extracted on
ice (5 min). The samples were centrifuged (10,000 × g,
5 min, 4 °C), and the supernatants were boiled with 0.33 volume of
SDS sample buffer. Protein concentrations were determined using the
Bradford method (36). Proteins (for whole heart extract, 100 µg
loaded/lane) were separated by SDS-polyacrylamide gel electrophoresis
on 10% (w/v) polyacrylamide gels that had been formed in the presence
of 0.5 mg/ml myelin basic protein (MBP) for the assay of ERKs, 0.5 mg/ml GST-c-Jun(1-135) for the assay of SAPKs/JNKs, or 0.5 mg/ml
GST-MAPKAPK2(46-400) for the assay of p38-MAPKs, with a 6% (w/v)
stacking gel. Following electrophoresis, SDS was removed from the gels
by washing in 20% (v/v) propan-2-ol in 50 mM Tris-HCl (pH
8.0) (3 × 20 min). The propan-2-ol was removed by washing in 50 mM Tris-HCl (pH 8.0), 5 mM 2-mercaptoethanol (3 × 20 min). Proteins were denatured in 6 M
guanidine-HCl, 50 mM Tris-HCl (pH 8.0), 5 mM
2-mercaptoethanol (2 × 30 min) and then renatured in 50 mM Tris-HCl (pH 8.0), 5 mM 2-mercaptoethanol, 0.04% (v/v) Tween 40 (1 × 30 min, 2 × 1 h, 1 × 18 h, 1 × 30 min, 4 °C). The gels were equilibrated to
room temperature with 40 mM Hepes (pH 8.0), 2 mM dithiothreitol, 10 mM MgCl2
(2 × 30 min) and incubated for 3 h with 12.5 µCi/gel
[
-32P]ATP in 5 ml of 40 mM Hepes (pH 8.0),
0.5 mM EGTA, 10 mM MgCl2, 50 µM ATP, 0.1 µM PKI. The reaction was
stopped, and gels were washed with 1% (w/v) disodium pyrophosphate,
5% (w/v) trichloroacetic acid. The gels were dried onto 3MM Whatman
chromatography paper and autoradiographed. In gel kinase activities
were quantified by laser scanning densitometry.
Western Blot Analysis-- Proteins (30 µl) were separated by SDS-polyacrylamide gel electrophoresis on 10% (w/v) polyacrylamide gels and transferred electrophoretically to nitrocellulose (42). Nonspecific binding sites were blocked with 5% (w/v) nonfat milk powder in 20 mM NaH2PO4, 80 mM Na2HPO4, 100 mM NaCl, 0.05% (v/v) Tween-20 (pH 7.5) (PBST) for 30 min, and the blots were incubated with JNK1 or p38-MAPK antibodies (1:100 dilution in blocking solution, overnight, 4 °C). After washing in PBST (3 × 5 min), the blots were incubated with horseradish peroxidase-linked anti-rabbit IgG antibodies (1:5000 dilution in PBST containing 1% (w/v) nonfat milk powder, 1 h, room temperature). The blots were washed again in PBST (3 × 5 min), and the bands were detected using ECL with exposure to Hyperfilm MP. Blots were quantified by laser scanning densitometry.
Fast Protein Liquid Chromatography of ERKs and
MAPKAPK2--
Supernatants of heart powders homogenized with 3 volumes
of buffer A were diluted 4-fold with buffer A and recentrifuged
(10,000 × g, 5 min, 4 °C). Proteins in samples (0.5 ml) were separated by fast protein liquid chromatography (FPLC). ERKs
were separated on a Mono Q HR5/5 column equilibrated with 50 mM Tris/HCl (pH 7.3), 2 mM EDTA, 2 mM EGTA, 0.1% (v/v) 2-mercaptoethanol, 5% (v/v) glycerol,
0.03% (v/v) Brij-35, 0.3 mM
Na3VO4, 1 mM benzamidine, and 4 µg/ml leupeptin. Following a 5-ml isocratic wash, ERKs were separated
using a linear NaCl gradient (20 ml, 0-0.33 M NaCl) at
flow rate of 1 ml/min with collection of 0.5-ml fractions. They were
assayed by the incorporation of 32P from
[-32P]ATP into MBP by the direct method as described
previously (43). Samples of fractions were also taken for in gel kinase
assays and were boiled with 0.33 volume of SDS sample buffer.
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RESULTS |
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Activation of "Stress-regulated" MAPKs by H2O2-- In gel kinase assays of hearts perfused for 30 min showed maximal activation of both p46 and p54 SAPKs/JNKs with 0.5 mM H2O2 (Fig. 1A). Activation was reduced at higher concentrations. This activation was never as great as that observed after ischemia (20 min) followed by reperfusion (10 min) (Fig. 1A). We also confirmed that ischemia alone (20 min) did not activate the SAPKs/JNKs (Fig. 1A). JNK1 antibodies immunoprecipitated all of the p46 SAPK/JNK activity (Fig. 1B, top panel) and protein (Fig. 1B, bottom panel), and approximately 25% of the p54 SAPK/JNK activity (Fig. 1B, top panel). It is not possible to determine the proportion of p54 SAPK/JNK protein immunoprecipitated by the JNK1 antibody (Fig. 1B, bottom panel) because of the interference by immunoglobulins in this region of the gel. These data confirm that the activities principally responsible for phosphorylation of c-Jun(1-135) in the in gel kinase assays (Fig. 1A) were SAPKs/JNKs.
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Activation of ERKs-- Activation of MAPKs by 0.5 mM H2O2 was mainly confined to the stress-regulated forms. In gel kinase assays showed minimal phosphorylation of the ERK substrate, MBP, by extracts of hearts perfused with H2O2 for 30 min (results not shown). However, because ERKs are generally activated at earlier times than the SR-MAPKs in heart (29-31), activation of ERKs by H2O2 was also assessed in hearts perfused for 5 min using both in gel kinase assays and following FPLC on Mono Q columns. As a positive control, the effects of phorbol 12-myristate 13-acetate (PMA, which powerfully activates ERKs in hearts (29, 43, 44)) were also studied. p42 and p44 ERKs eluted from the Mono Q column at 0.22 M and 0.26 M NaCl, respectively, consistent with published data (29, 43, 44) and were activated in hearts perfused with PMA (1 µM, 5 min) (Fig. 3, A and B). Relatively little activation of p42 and p44 ERKs (ERK2 and ERK1 respectively) was observed after perfusion with H2O2 (Fig. 3, A and B). The identities of the kinases detected after FPLC were confirmed as p42 and p44 ERKs by in gel kinase assays with MBP as substrate (Fig. 3B).
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Activation of MAPKAPK2-- MAPKAPK2 is an established substrate of p38-MAPK (18). The activation of MAPKAPK2 was studied following FPLC on Mono S columns. Ischemia (Fig. 4A), ischemia/reperfusion (Fig. 4B), and H2O2 (Fig. 4C) all induced activation of MAPKAPK2. The effects of ischemia and H2O2 were comparable, but the effects of ischemia/reperfusion were consistently greater. SB203580 is a selective inhibitor for p38-MAPK and inclusion of 10 µM SB203580 (in Me2SO, 0.1% (v/v), 14 mM final concentration) in the perfusion media abolished the activation of MAPKAPK2 by all of these interventions (Fig. 4, A-C). To ensure that these effects were independent of Me2SO, 14 mM Me2SO was included in all perfusions. Independently, we showed that inclusion of this concentration of Me2SO had no effect on the activation of p38-MAPK or MAPKAPK2 by ischemia or ischemia/reperfusion as compared with perfusions in the absence of Me2SO (results not shown).
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Activation of SR-MAPKs and MAPKAPK2 by High Pressure Perfusion-- Hearts were perfused at 20 kilopascals (140 mm Hg) for 20 min to simulate aortic hypertension. p46 and p54 SAPKs/JNKs (Fig. 5A, top panel) and p38-MAPK (Fig. 5A, bottom panel) were activated. Activation of MAPKAPK2 was also detected (Fig. 5B).
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Effects of Free Radical Scavengers on the Activation of SR-MAPKs and MAPKAPK2 by Ischemia and Ischemia/Reperfusion-- To demonstrate that ROS and other free radicals are involved in the activation of SR-MAPKs and MAPKAPK2 by ischemia and/or ischemia/reperfusion, hearts were perfused with the OH· scavenger, Me2SO (0.4% (v/v), 56 mM), or with the lipophilic spin trap radical scavenger, BPN (10 mM, added in Me2SO (56 mM final concentration)). Perfusion of hearts with Me2SO or BPN/Me2SO under control conditions (no interruption in coronary flow) did not affect the activity of either p38-MAPK or SAPKs/JNKs. The activation of p38-MAPK by ischemia was unaffected by the presence of Me2SO but was greatly reduced by BPN/Me2SO (Fig. 6A). Consistent with this, the activation of MAPKAPK2 by ischemia was not reduced (and may even be increased) in the presence of Me2SO, whereas BPN essentially abolished the activation of MAPKAPK2 by ischemia (Fig. 7). In contrast, the activation of SAPKs/JNKs (Fig. 6B, upper panel), p38-MAPK (Fig. 6B, lower panel) and MAPKAPK2 (Fig. 7) by ischemia/reperfusion was essentially completely inhibited by 56 mM Me2SO.
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DISCUSSION |
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Global ischemia stimulates p38-MAPK and MAPKAPK2 activities in the
perfused heart (32, 33). On reperfusion of ischemic hearts, the
stimulation of p38-MAPK and MAPKAPK2 are maintained or increased (32,
33). In addition, activities of SAPKs/JNKs are stimulated by
ischemia/reperfusion (32-34). A variety of potentially cytotoxic
activators of the SR-MAPKs are produced by the heart in response to
ischemia and reperfusion. However, it is not clear which agent(s) is
responsible for the activation of these kinases. It was recognized some
years ago that there was a significant release of ROS under these
conditions (reviewed in Refs. 1, 2, and 4). Thus increases in
H2O2 (45), OH· (46, 47), and O2
(48, 49) have been detected in ischemia and in reperfusion following
ischemia. These species then induce the production of other radicals
(e.g. alkoxy and alkyl radicals) by reaction with membrane
lipids (50). Here, we have assessed the potential of ROS, as
exemplified by H2O2, to activate the SR-MAPKs
in perfused heart and tested whether the production of ROS and free
radicals may be responsible for the activation of these kinases by
ischemia and ischemia/reperfusion.
Activation of SR-MAPKs and MAPKAPK2 by H2O2-- Perfusion of rat hearts with H2O2 stimulated SAPKs/JNKs (Fig. 1, A and B) and p38-MAPK (Fig. 2, A and C). These results contrast with those of Knight and Buxton (34), who failed to detect activation of SAPKs/JNKs in hearts perfused with 0.5 mM H2O2 (34). Activation of both SAPKs/JNKs and p38-MAPK appeared to be critically dependent on the concentration of H2O2 because there was minimal activation at 0.1-0.2 mM, but with 0.5 mM H2O2 activation was maximal (Figs. 1A and 2A). At higher concentrations (1 mM H2O2), SAPK/JNK activity declined (Fig. 1A), but p38-MAPK activity was maintained. A similar activation pattern of the SAPKs/JNKs has been noted in astrocytes exposed to H2O2 (51). The activation of p38-MAPK by 0.5-1 mM H2O2 was comparable with the activation seen after ischemia and ischemia/reperfusion (Fig. 2A). These data suggest that increases in H2O2 concentrations (or other ROS) in the heart during ischemia and ischemia/reperfusion (45) could play a role in activation of p38-MAPK. In contrast to the activation of p38-MAPK, the activation of SAPKs/JNKs by 0.5 mM H2O2 was greater than that seen after ischemia but less than that after ischemia/reperfusion (Fig. 1A). This suggests that factors other than ROS may be involved in the activation of SAPKs/JNKs during ischemia/reperfusion.
The species of ROS responsible for the activation of the SR-MAPKs by H2O2 is not clear. Exposure of isolated hearts or ventricular myocytes to H2O2 leads to an iron-dependent formation of OH· (52-54). Although OH· may be partly responsible for some of the cardiotoxic effects of H2O2, OH·-independent effects of H2O2 have also been detected (55). At low concentrations (14 mM), the OH· scavenger Me2SO did not significantly inhibit the activation of p38-MAPK or MAPKAPK2 (an index of p38-MAPK activation) by 0.5 mM H2O2 (results not shown). Further experiments examining the effects of higher concentrations of Me2SO on the activation of SR-MAPKs by H2O2 are indicated. Several isoforms of the SAPKs/JNKs have been identified by molecular cloning (56). At least three genes produce alternatively spliced transcripts encoding proteins of approximately 46 and 54 kDa (56). An antibody to human JNK1 immunoprecipitated all of the p46 SAPK/JNK activity and approximately 25% of the p54 SAPK/JNK activity stimulated by ischemia, ischemia/reperfusion, or H2O2 in perfused heart (Fig. 1B, top panel). This suggests that there is activation of at least one other isoform of p54 SAPK/JNK. Although antibodies to JNK2 detect a protein in neonatal rat ventricular myocytes, we found that these antibodies were not suitable for immunoprecipitation (results not shown). SB203580 is a selective inhibitor of p38-MAPK (17, 57). The 38-kDa MAPK activity detected using in gel assays with GST-MAPKAPK2(46-400) as a substrate was completely inhibited by 10 µM SB203580 (Fig. 2B), indicating that this activity is indeed attributable to p38-MAPK. However, an antibody to the C terminus of murine p38-MAPK immunoprecipitated only approximately 50% of the activity (Fig. 2C, top panel). The immunoprecipitated form of p38-MAPK migrated slightly more slowly than the residual activity in the supernatant (Fig. 2C, top panel), suggesting that more than one isoform of p38-MAPK is activated by ischemia, ischemia/reperfusion, or H2O2 in perfused heart. Using the same antibody for Western blots, bands of approximately 38 kDa were detected in both the immunoprecipitates and the residual supernatants (Fig. 2C, bottom panel), although, consistent with the p38-MAPK activity data (Fig. 2C, top panel), the band in the immunoprecipitates migrated more slowly than the band in the supernatants (Fig. 2C, bottom panel). This suggests that although the antibody is more selective for a particular isoform(s) of p38-MAPK in the native form, it also detects other forms after denaturation. At least five isoforms of p38-MAPKs have been identified: p38-MAPK (15, 16, 18) of which there are two alternatively spliced isoforms (17), p38-MAPKActivation of ERKs by
H2O2--
H2O2
activates the ERKs in HeLa, Rat1, NIH 3T3, and PC12 cell lines (62) and
in primary cultures of rat astrocytes (51). There is disagreement about
whether H2O2 does (62) or does not (63) produce
a significant activation of ERKs in primary cultures of vascular smooth
muscle cells. In the study in which no ERK activation was detected
(63), there was significant activation of ERKs by another ROS, namely
O2. We detected only minimal ERK1 and ERK2 activation in
hearts perfused with 0.5 mM H2O2
for 5 min, although there was significant activation in hearts perfused with 1 µM PMA (Fig. 3, A and B).
There was no detectable activation of ERKs after 30 min of perfusion
with 0.5 mM H2O2 (results not shown). These data contrast with a previous study that showed activation of ERKs rather than SAPKs/JNKs in hearts perfused with H2O2 (34). However, in this study (34), ERKs
were partially purified by batch elution from DEAE-Sephacel with 0.5 M NaCl and assayed with a tetrapeptide derived from MBP.
Such an elution protocol would elute all MAPK species and is therefore
less specific than separation on Mono Q FPLC using a NaCl gradient,
which was used here.
Mechanisms of Activation of SR-MAPKs and MAPKAPK2 during Ischemia
and Ischemia/Reperfusion--
As discussed above,
H2O2 generates free radical ROS in the heart
and activates SR-MAPKs (Figs. 1 and 2) and MAPKAPK2 (Fig. 4). Equally,
ROS are generated in the heart during ischemia and ischemia/reperfusion. We therefore sought evidence that ROS and other
free radicals mediate the activation of SR-MAPKs in ischemia and
ischemia/reperfusion. We used two different free radical scavengers, the OH· radical scavenger Me2SO and the lipophilic
spin trap BPN. Both have been found to be cardioprotective under
situations of increased oxidative stress (54, 64). At a concentration
of 10 mM BPN/56 mM Me2SO (solvent
carry-over), activation of p38-MAPK and MAPKAPK2 by ischemia was
inhibited (Figs. 6A and 7). In contrast, 56 mM Me2SO alone inhibited activation of SR-MAPKs and MAPKAPK2
after ischemia/reperfusion (Fig. 6B and 7). These data
implicate ROS and free radicals in the activation of SR-MAPKs during
ischemia and ischemia/reperfusion but suggest that different radicals
may be involved during the ischemic and reperfusion phases. It has recently been suggested that O2 and
H2O2 are formed during simulated ischemia in
isolated cardiac myocytes, whereas OH· and further
H2O2 are generated during simulated reperfusion
(65). This is entirely consistent with our data (Figs. 6 and 7).
Activation of SR-MAPKs and MAPKAPK2 by High Pressure Perfusion-- Using in gel kinase assays with MBP as a substrate, we have previously shown that short term perfusion (5 min) of hearts at high aortic pressures activates ERKs (44). Furthermore, the upstream activators of the ERKs, the MAPK (or ERK) kinase group of MAPK kinases, were also activated (44). This work was completed prior to the identification of the SR-MAPKs. Here, we show that perfusion of hearts at high aortic pressure for longer times (20 min) additionally stimulated the activities of SAPKs/JNKs, p38-MAPK, and MAPKAPK2 (c.f. ischemia, ischemia/reperfusion, and H2O2). Although the mechanism of activation of the SR-MAPKs by high pressure perfusion is unclear, it could represent ischemia/reperfusion effects, because hypertension in vivo is known to induce subendomyocardial ischemia (reviewed in Ref. 3).
Significance of SR-MAPKs in the Heart--
The biological
consequences of activation of SR-MAPKs in the heart are poorly
understood. Activation of the SAPKs/JNKs would be expected to result in
phosphorylation of the c-Jun and ATF2 transcription factors, increasing
their trans-activating activity (reviewed in Ref. 6). In
this regard, we have shown that hyperosmotic stress activates SAPK/JNKs
in ventricular myocytes (31), and c-Jun and ATF2 become phosphorylated
(66). Transcription of c-jun is regulated by a number of
cis-acting regulatory sequences in the c-jun
promoter region, including two sites (jun1 and
jun2) that bind c-Jun/ATF2 heterodimers. Thus activation of
SAPKs/JNKs is potentially able to up-regulate c-jun
expression. Increased expression of c-jun occurs during
ischemia/reperfusion in isolated hearts (67) and during hypoxia in
cultured myocytes (68). Furthermore, H2O2
induces c-jun expression in NIH 3T3 cells (69). Thus
SAPK/JNK-dependent activation of c-Jun might be expected to
increase gene expression and anabolism. Indeed, SAPKs/JNKs have been
proposed to be mediators of the 1-adrenergic stimulation of hypertrophic growth in the ventricular myocyte (70). However, activation of SAPKs/JNKs and c-Jun (71) induces apoptosis in a number
of cell lines, which may be particularly pertinent in the ischemic
heart. The p38-MAPKs have also been implicated in
1-adrenergic stimulation of hypertrophic growth in the
ventricular myocyte (72). One of their substrates (MAPKAPK2)
phosphorylates the small heat shock proteins Hsp25/27, which may be
cytoprotective (26). However, activation of p38-MAPK may be apoptotic
(73). The ultimate biological effects of activation of SR-MAPKs may depend on the duration and extent of their activation (74).
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FOOTNOTES |
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* This work was supported by grants from the British Heart Foundation.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
¶ British Heart Foundation Lecturer in Basic Science.
To whom correspondence should be addressed: NHLI Div.,
Imperial College School of Medicine, Dovehouse St., London SW3 6LY, UK.
Tel.: 44-171-352-8121 (Ext. 3306/3314); Fax: 44-171-823-3392; E-mail:
p.sugden{at}ic.ac.uk.
1
The abbreviations used are: ROS, reactive oxygen
species; BPN, N-t-butyl--phenyl nitrone; ERK,
extracellular signal-regulated kinase; FPLC, fast protein liquid
chromatography; GST, glutathione S-transferase; MAPK,
mitogen-activated protein kinase; MAPKAPK, MAPK-activated protein
kinase; MBP, myelin basic protein; PKI, cyclic
AMP-dependent protein kinase inhibitory peptide; SAPK, stress-activated protein kinase; JNK, c-Jun N-terminal kinase; SR-MAPKs, stress-regulated MAPKs (SAPKs/JNKs and p38-MAPKs).
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