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FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online September 17, 2001 as doi:10.1096/fj.01-0353fje. |
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Physiologisches Institut, Justus-Liebig-Universität Giessen, Germany
2Correspondence: Physiologisches Institut, Justus-Liebig-Universität, Aulweg 129, 35392 Giessen, Germany. E-mail: Gerhild.Taimor{at}physiologie.med.uni-giessen.de
SPECIFIC AIMS
It was the aim of the present study to characterize molecular steps of apoptosis induction by nitric oxide (NO) in ventricular cardiomyocytes of the rat. We focused on members of the mitogen-activated protein kinase (MAPK) family and their downstream target, the transcription factor AP-1.
PRINCIPAL FINDINGS
1. NO induces apoptosis via AP-1 activation in cardiomyocytes
To test whether NO activates the transcription factor AP-1 in cardiomyocytes, binding activity of AP-1 in nuclear extracts from these cells was analyzed in retardation assays using a radioactive labeled oligonucleotide, which contained the specific binding motif of AP-1 (TRE). Addition of SNAP (100 µM) to cardiomyocytes increased transiently the AP-1 binding activity (Fig. 1
, lanes 1–6). This binding was specific since it could be blocked by preincubation with an excess amount of unlabeled TRE oligonucleotides (lanes 7–9), but not with oligonucleotides containing a mutated TRE site (lanes 10, 11). 2 h after addition of SNAP, AP-1 binding activity was increased to 433.6 ± 122.3% vs. controls (n=5, P<0.05). The induction of AP-1 binding activity by SNAP was dose-dependent. Only doses of 10 and 100 µM SNAP, that were shown before to induce apoptosis, increased AP-1 activity significantly. AP-1 activity could be scavenged intracellularly with decoy oligonucleotides. Preincubation of cardiomyocytes with CRE decoy oligonucleotides for 5 h, but not with scrambled CRE oligonucleotides, inhibited AP-1 binding activity in SNAP-induced cardiomyocytes.
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The concentration of SNAP found to activate AP-1 also induced apoptosis in cardiomyocytes. As determined by chromatin condensation, 24 h after addition of SNAP (100 µM) apoptosis was observed in 10.5 ± 0.7% of cardiomyocytes vs. 4.9 ± 0.8% in controls (n=4, P<0.05) (Fig. 2
). Intracellular scavenging of AP-1 by preincubation of cardiomyocytes with TRE or CRE decoy oligonucleotides totally abolished this apoptotic response (3.8±0.5% and 4.4±0.5% apoptotic cells, respectively). The scrambled CRE oligonucleotide, which was unable to block AP-1 binding activity, did not inhibit SNAP-induced apoptosis (14.5±0.8% apoptotic cells, n=4, P<0.05 vs. control).
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2. JNK is involved in AP-1 activation and apoptosis induction
AP-1 is a prominent target of JNK. Therefore, we tested whether JNK is involved in NO-induced AP-1 activation and apoptosis induction. Analysis of the phosphorylation state of JNK in cardiomyocytes using a phospho-specific JNK antibody revealed activation of JNK by SNAP (100 µM) with a maximum increase of 87.0 ± 27.3% after 30 min (n=7, P<0.05), slowly declining thereafter. A pharmacological activator of JNK1, Ro318220 (10 µM), which increased JNK activity by maximal 83.0 ± 24.0% after 1 h, also induced AP-1 binding activity in cardiomyocytes and increased the number of apoptotic cardiomyocytes to 18.7 ± 1.2% (n=4, P<0.05 vs. control). This apoptosis induction was abolished by preincubation of cardiomyocytes with TRE or CRE decoy oligonucleotides. For control reasons, we tested whether ERK might be involved in apoptosis induction by Ro318220. Specific inhibition of the MEK/ERK pathway by PD98059 (10 µM) did not decrease Ro318220-induced apoptosis. The findings demonstrate that activation of JNK in cardiomyocytes increases AP-1 activity and induces apoptosis via this pathway. This pathway is also involved in NO-induced apoptosis.
3. ERK is involved in AP-1 activation and apoptosis induction
In Western blot analysis using ERK2 antibodies increased amounts of the phosphorylated, active form of ERK2 were detected after incubation with SNAP (100 µM). Inhibition of this ERK activation with the MEK1 inhibitor PD98059 (10 µM) reduced the number of apoptotic cells from 20.1 ± 1.8% in SNAP-induced cultures to 13.2 ± 1.2% in cultures preincubated with PD98059 (n=6, P<0.05). To analyze when ERK modulates the NO-induced apoptotic signaling cascade, we first investigated the influence of ERK inhibition by PD98059 on JNK activation. Addition of PD98059 (10 µM) did not inhibit SNAP-induced activation of JNK (237±51%, n=4, P<0.05 vs. control). This indicates that ERK interferes with steps downstream of JNK. We found that incubation with PD98059 blocked activation of AP-1 by SNAP.
4. p38 MAPK does not mediate NO-induced apoptosis
In Western blot analysis using phospho-specific p38 MAPK antibodies, a basal amount of active p38 was found. This p38 MAPK activity was not increased by incubation with SNAP, but was abolished by incubation with SB202190 (10 µM). Incubation of cardiomyocytes with SB202190 did not inhibit apoptosis induction by SNAP (17.9±0.9% apoptotic cells, n=3, P<0.05 vs. control). Instead, SB202190 increased the percentage of apoptotic cardiomyocytes to 19.9 ± 0.7% (n=3, P<0.05 vs. control). This indicates that p38 MAPK exerts an antiapoptotic action under basal culture conditions.
CONCLUSIONS
The main result of this study is that induction of apoptosis by NO in adult cardiomyocytes from rat is mediated via an activation of the transcription factor AP-1. Upstream of AP-1, activation of JNK and MEK/ERK are involved (summarized in Fig. 3
).
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Involvement of AP-1 in NO-induced apoptosis was demonstrated by inhibition of this process with decoy oligonucleotides, which inhibited AP-1 binding activity. Because JNK is known to cause the formation of the active AP-1 complex, we analyzed whether JNK activation would also provoke apoptosis in adult cardiomyocytes. A pharmacological activator of JNK, Ro318220, activated both, JNK and AP-1, in these cells and it also increased the number of apoptotic cardiomyocytes. This induction of apoptosis by activation of JNK could be abolished using decoy oligonucleotides containing AP-1 binding sites. The results show that JNK activation induces apoptosis via AP-1. NO also activates JNK in adult cardiomyocytes. Together with results from the Ro318220 experiments, this analysis implies that JNK activation represents an intermediate step in NO-induced AP-1 activation (Fig. 3)
.
NO activated ERK in the cardiomyocytes. We used the MEK1 inhibitor PD98059 to block this pathway. Presence of PD98059 abolished NO-induced apoptosis. It also inhibited the NO-induced activation of AP-1. The results, therefore, confirm a key role of AP-1 in the induction of apoptosis by NO. Further experiments were performed to decide whether MEK/ERK activation is needed in parallel or downstream to JNK activation in this apoptotic mechanism. PD98059 did not inhibit JNK activation by NO. This places the MEK/ERK pathway in parallel to JNK in NO-induced apoptosis as illustrated in Fig. 3
.
Another member of the MAPK family, p38 MAPK, was also analyzed. We did not find a p38 MAPK activation in presence of NO. Neither did we observe that addition of the inhibitor of p38 MAPK, SB202190, blocked the induction of apoptosis by NO. In fact, presence of SB202190 had an opposite effect: In the absence of NO, SB202190 increased the amount of apoptotic cells. This suggests that in these adult cardiomyocytes p38 MAPK exerts an antiapoptotic role. A similar protective effect of p38 kinase has been shown for the case of ß-adrenergic receptor-stimulated apoptosis in adult cardiomyocytes .
The present study shows that in adult cardiomyocytes from the rat, induction of apoptosis by NO involves activation of JNK, MEK/ERK, and AP-1. The findings on JNK are in apparent contrast to a recent study of neonatal cardiomyocytes from rat. In that cell model, JNK activation was found to reduce NO-induced apoptosis. Inhibition of the MEK/ERK pathway by PD98059 had no effect on NO-induced apoptosis. These differences may be due to biological differences between the investigated cell types. It reminds of the findings in neuronal cells, where c-jun has opposite effects on apoptosis at different stages of differentiation. Similarly, the different roles of JNK in neonatal and adult myocytes may be due to differences in the phenotypical differentiation.
The role of ERK in apoptotic processes may also vary among cell types. The kinetics of ERK-activation might be of critical importance. Sustained activation of ERK seems related to inhibition of apoptosis as found in neuronal cells after NGF withdrawal. In contrast, transient activation of ERK induces apoptosis as shown in the present study of cardiomyocytes and in another study of mesangial cells exposed to H2O2. In the latter study, such a transient apoptosis-inducing ERK activation was associated with an increase in c-fos expression, which also suggests AP-1 involvement.
In the present study, we identified the JNK/AP-1-pathway as a key element of NO-induced apoptosis in adult cardiomyocytes. NO-mediated apoptosis in adult cardiomyocytes might occur in inflammatory processes and upon ischemia-reperfusion in the ventricular myocardium. Our results identify JNK and AP-1 as targets for antiapoptotic therapy in these cardiac disease states. This central role of AP-1 in NO-induced apoptosis in adult cardiomyocytes also raises questions about the downstream targets of AP-1 responsible for the apoptotic effect. TRE and CRE elements are found in various promoters, and identification of the targets of AP-1 related to NO-induced apoptosis in cardiomyocytes awaits further investigation.
FOOTNOTES
1 To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.01-0353fje; to cite this article, use FASEB J. (September 17, 2001) 10.1096/fj.01-0353fje 
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