© 2002 by European Society of Cardiology
Copyright © 2002, European Society of Cardiology
Molecular mechanisms of non-apoptosis by Fas stimulation alone versus apoptosis with an additional actinomycin D in cultured cardiomyocytes
aSecond Department of Internal Medicine, Gifu University School of Medicine, 40 Tsukasa-Machi, Gifu 500-8705, Japan
bDepartment of Cardiovascular Regeneration Science, Gifu University School of Medicine, 40 Tsukasa-Machi, Gifu 500-8705, Japan
* Corresponding author. Tel.: +81-58-267-2607; fax: +81-58-267-2933 gifuim-gif{at}umin.ac.jp
Received 17 December 2001; accepted 9 May 2002
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Abstract |
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 Top Abstract 1. Introduction2. Methods3. Results4. DiscussionReferences |
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Objective: In cultured cardiomyocytes, apoptosis is induced not by Fas stimulation, a popular inducer of apoptosis, but by an additional treatment with actinomycin D (AD), a transcription inhibitor, although the mechanism is unknown. Our hypothesis is that Fas stimulation not only activates pro-apoptotic signals but also may inhibit some of them, and this inhibition is blocked by AD. Methods: Cultured neonatal mouse cardiomyocytes were treated with agonistic anti-Fas antibody (FA), AD, or both (FA+AD). In this system, apoptotic signals related to Fas-induced apoptotic pathways were examined by RT-PCR and immunoblotting. In addition, antisense oligonucleotide (AS) studies were carried out. Results: The treatment with FA+AD induced up-regulation of Fas, activation of c-Jun N-terminal kinase (JNK), which is one of the key molecules of the alternate pathway of Fas-induced apoptosis, up-regulation of Bax, up-regulation and activation of caspase-3, activation of caspase-3-dependent DNase (CAD), and final DNA fragmentation and apoptotic morphologies in cardiomyocytes. FA alone or AD alone did not affect any part of the above pathway. However, mRNA of mitogen-activated protein kinase phosphatase-1 (MKP-1), an inactivator of JNK, was up-regulated by FA alone, but not by FA+AD or AD alone. Pretreatment with AS against MKP-1 induced apoptosis in FA alone-treated cardiomyocytes, whereas AS against JNK1 prevented apoptosis induced by FA+AD. On the other hand, FA+AD did not result in the activation of either caspase-8, one of the key molecules of the classic pathway in Fas-induced apoptosis, p38 MAPK, or extracellular signal-regulated kinase (ERK). Conclusions: Cardiomyocyte apoptosis by FA+AD depends on the alternate pathway through the JNK, Bax and caspase-3, and CAD-dependent pathways including a positive feedback mechanism of Fas up-regulation. The molecular mechanism that prevents Fas stimulation alone from inducing apoptosis involves up-regulation of MKP-1, an inhibitor of JNK; this up-regulation is inhibited by AD.
KEYWORDS Apoptosis; Cell culture/isolation; Gene expression; Myocytes; Signal transduction
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1. Introduction |
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TopAbstract1. Introduction2. Methods3. Results4. DiscussionReferences |
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Fas (APO-1/CD95) is a widely expressed cell death receptor that has a critical role in the regulation of the immune system, tissue homeostasis and apoptosis in various cell types [13]. Fas action is complicated in its cell specificity. In some cell types, Fas stimulation alone with agonistic Fas antibody or soluble Fas ligand is adequate for induction of apoptosis, while a combined use of an RNA transcription inhibitor such as actinomycin D (AD), protein synthesis inhibitor such as cycloheximide, or other cytotoxic agents is necessary for execution of apoptosis in several kinds of cells such as hepatocytes, mesangeal cells, and vascular smooth muscle cells in culture [4,14,24]. Cardiomyocytes belong to the latter category [22]. Recently, we also reported that in cultured neonatal and adult cardiomyocytes, apoptosis was induced not by Fas stimulation (FA) but by a combined treatment with actinomycin D (FA+AD) [12,18]. However, the molecular mechanism has not been examined until now, although the presence has long been postulated of unknown protein(s) inhibiting the Fas-induced apoptotic signal transduction. A similar phenomenon was observed in the case of tumor necrosis factor (TNF)-
-induced apoptosis in several kinds of cells such as mesangeal cells where an additional treatment with AD was necessary for execution of apoptosis [5,11]. In this case, a recent study by Guo et al. [5] unraveled one of the responsible proteins, mitogen-activated protein kinase phosphatase-1 (MKP-1), which is one of the dual-specificity phosphatases that selectively inactivate tyrosine-phosphorylated mitogen-activated protein kinases (MAPKs) through dephosphorylation [6,21]. They proposed that a TNF--induced MPK-1 suppressed a prolonged activation of c-Jun N-terminal kinase (JNK), an apoptosis inducer through activation of transcription factors such as AP-1 and other cellular targets [1,7], in mesangeal cells [5]. Thus, it is suggested that MAPK family and the related proteins may be associated with molecular mechanisms of apoptosis induced by FA+AD and non-apoptosis by FA alone in cardiomyocytes. In the present study, to define the molecular mechanisms of apoptosis by FA+AD and non-apoptosis by FA alone in cardiomyocytes, we examined JNK, p38 MAPK, extracellular signal-regulated kinase (ERK), and MKP-1. In addition, we examined the other signaling pathways downstream of Fas, such as the Bcl-2 family, caspase-3 and -8, caspase-3 activated DNase (CAD) and inhibitor of CAD (ICAD), which have not been well elucidated in cardiomyocyte apoptosis.
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2. Methods |
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TopAbstract1. Introduction2. Methods3. Results4. DiscussionReferences |
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The present investigation conforms with the Guide for the Care and Use of Laboratory Animals published by the US National Institute of Health (NIH Publication No. 85-23, revised 1996).
2.1 Culture and treatments of cardiomyocytes
Cardiomyocytes were isolated from neonatal (1- to 2-day-old) Balb/c mice as previously reported [18]. Cardiomyocytes were plated at a density of 5x104 cells/cm2 in tissue culture plates. Cardiomyocytes were incubated overnight in D-Valine MEM (Gibco) containing 5% fetal bovine serum (Gibco). On the next day, vehicle (0.01 mol/l phosphate buffered saline, PBS), 1 µg/ml anti-Fas antibody (FA; clone Jo2, Pharmingen), 0.05 µg/ml actinomycin D (AD; Wako Pure Chemical Industry), or both (FA+AD) were added. The present apoptosis inducing system was also intervened by vanadate (500 µM, Wako), a protein-tyrosine-phosphatase inhibitor.
2.2 Cell viability
Viable cells were counted on 3-cm dishes using trypan blue staining. Experiments were carried out in triplicate in each group.
2.3 RNA extraction and Northern blotting
The acid guanidium–phenol–chloroform method was used to extract total RNA from cardiomyocytes that had been stimulated for 3 h, and 8 µg of RNA was electrophoresed on 1.1% agarose gels containing formaldehyde. RNA was then transferred to nylon membranes. The membranes were hybridized with a whole coding region of cDNA of Fas, Bcl-2, Bax, caspase-3, ICAD, and MKP-1 labeled with [-32P]dCTP (NEN). Hybridization was performed in 50 mM Tris–HCl (pH 7.5), 0.1% SDS, 1 M NaCl, 10% dextran sulfate, and 200 µg/ml yeast tRNA for 18 h at 65 °C. After washing at 60 °C with 2xSSC containing 0.1% SDS, the membranes were autoradiographed and signals were quantified. Quantitative analyses of mRNA were normalized to the GAPDH mRNA content or ribosomal RNA content.
2.4 RT-PCR
Total RNA (1 µg), taken from the cardiomyocytes, was reverse-transcribed into cDNA and subsequently the synthesized cDNA was amplified. CAD mRNA expression was quantified by standard calibrated reverse transcription PCR. A 100-bp CAD cDNA fragment was amplified using the forward primer (5'-TGAGCTCCTATTGCTCACCG-3') and reverse primer (5'-TATTCTGGCTCACGTGATGC-3') at 36 cycles of 1 min at 94 °C, 1 min at 53 °C, and 1 min at 72 °C each from total RNA of neonatal cardiomyocytes. These RT-PCR products were electrophoresed on 2% agarose gel containing ethidium bromide and the quantities were analyzed by densitometry.
2.5 Western blotting
Cardiomyocytes were stimulated for 6 h, then washed twice with PBS and lysed in Laemmli's buffer. The amount of protein in each lysate was determined using a BCA protein assay kit (Pierce). The samples (5 µg) were heated in a boiling water bath for 3 min and subjected to electrophoresis on a 10% polyacrylamide–SDS slab gel. Proteins were transferred onto a nylon membrane following electrophoresis in 25 mM Tris–HCl, 192 mM glycine, and 0.1% SDS. The membrane was blocked with 5% non-fat dry milk overnight at 4 °C, then incubated with the primary antibody against Fas, Bcl-2, Bax, caspase-3, caspase-8 (Santa Cruz), CAD, ICAD (Millennium Biotechnology), phospho-c-Jun II (Ser63), phospho-p38 MAPK, phospho-ERK, c-Jun, p38 MAPK, or ERK (New England Biolabs) in PBS containing 0.05% (v/v) Tween 20 (TPBS) at room temperature for 60 min. The immunoreactive protein bands were then visualized using biotinylated secondary antibody, a peroxidase-conjugated avidin–biotin complex (Vector), and a Konica Immunostain kit (Konica). The signals were quantified by densitometry.
2.6 Antisense oligonucleotide transfection
Cardiomyocytes were transfected with 0.33, 1, and 3.3 µM MKP-1 antisense (AS) or sense (S) oligonucleotide (phosphorothioate-modified and FITC-labeled) in Effectene Transfection Reagent (Qiagen) for 24 h. Oligonucleotide sequences (20 nt) were as follows: MKP-1-AS, 5'-GGAACTCAGTGGAACTCAGG-3'; MKP-1-S, 5'-CCTGAGTTCCACTGAGTTCC-3'. This AS has been well characterized and these concentrations were reported to effectively suppress MKP-1 activity [1]. Then the medium was changed and the cells were treated with 1 µg/ml FA. Twenty-four hours later, the viability of the cardiomyocytes was checked by trypan blue dye exclusion method, DNA fragmentation was detected by in situ nick end-labeling (TUNEL) and DNA gel electrophoresis, and the ultrastructure was examined under a transmission electron microscope as previously reported [18]. JNK1-AS or JNK1-S oligonucleotide (phosphorothioate-modified and FITC-labeled) transfection was carried out to the cardiomyocytes, at concentrations of 0.33, 1, and 3.3 µM, by the same method of transfection of MKP-1-AS and MKP-1-S oligonucleotides. Oligonucleotide sequences (24 nt) were as follows: JNK1-AS, 5'-ATCATGAGCAGAAGCAAGCGAGAC-3'; JNK1-S, 5'-GTCACGCTTGCTTCTGCTCATGAT-3' [7]. The cells were then treated with FA+AD. Cell viability, DNA fragmentation, and the ultrastructure of the cells were examined 24 h later.
2.7 Statistical analysis
All experiments were done in triplicate and values were expressed as the mean±S.E.M. To test the differences between experimental groups, a Student's t-test or ANOVA was carried out followed by Newman–Keul's multiple comparison test. A value of P<0.05 was chosen as the limit of statistical significance.
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3. Results |
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TopAbstract1. Introduction2. Methods3. Results4. DiscussionReferences |
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As previously shown, the cardiomyocytes presented typical apoptotic ultrastructure and internucleosomal DNA fragmentation as early as 3 h after the addition of a mixture of 1 µg/ml FA and 0.05 µg/ml AD [18]. In addition, the viability of the cells evaluated by the dye-exclusion method decreased 6 h later. Vehicle (PBS), FA, or AD alone did not cause apoptosis in the cardiomyocytes during incubation up to 24 h.
3.1 JNK is activated in Fas-induced cardiomyocyte apoptosis
In the cardiomyocytes exposed to FA alone, c-Jun was not phosphorylated during 3 h. However, phosphorylation of c-Jun, indicating JNK activation, was detected 30 min and later after the treatment with additional AD (Fig. 1). When vanadate, a protein-tyrosine-phosphatase inhibitor, was substituted for AD, phosphorylation of c-Jun was similarly detected. Therefore, it is suggested that FA up-regulates a factor that inhibits JNK activity, such as phosphatases like MKP-1 (see Section 3.2).
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We also examined phosphorylation of ERK and p38 MAPK by Western blotting, but failed to detect phosphorylated forms of these MAPKs by any treatments including FA+AD (data not shown).
3.2 Fas stimulation up-regulates mRNA of MKP-1
We next examined MKP-1 expression in the present model. MKP-1 is one of the phosphatases that selectively inactivates MAPKs through dephosphorylation [6,21]. As shown in Fig. 2, the treatment with FA alone enhanced mRNA of MKP-1 in a time-dependent manner, whereas the addition of AD significantly suppressed its enhancement.
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3.3 Treatments with antisense oligonucleotide against MKP-1 and JNK modulate execution of Fas-induced apoptosis of cardiomyocytes
For more definitive evidence of the critical roles of MKP-1 and JNK in the execution of Fas-stimulated apoptosis of cardiomyocytes, we interrupted their function by transfection with antisense oligonucleotide against MKP-1 (MKP-1-AS) or with that against JNK1 (JNK1-AS). Sense oligonucleotides (MKP-1-S and JNK-1-S) were used as controls. Successful transfection of oligonucleotides into the cardiomyocytes was confirmed under a fluorescent microscope. MKP-1-AS-transfected cells lost significant viability during 24 h by the treatment with FA alone, while untransfected control cells were almost intact and significantly fewer MKP-1-S-transfected cells were dead (Fig. 3A). A DNA ladder pattern was noted and typical apoptotic figures were ultrastructurally observed in the MKP-1-AS transfected cardiomyocytes after treatment with FA alone (Figs. 3B and 4A–C). That is, MKP-1-AS dose-dependently induced apoptotic death of cardiomyocytes in FA alone. When subsequently treated with FA+AD for 24 h, the viability of JNK1-AS-transfected cardiomyocytes was significantly improved in an AS dose-dependent manner, compared with that of JNK1-S-transfected and untransfected cells (Fig. 3A). A DNA ladder pattern disappeared and apoptotic figures were not noted in cardiomyocytes transfected with a higher dose of JNK1-AS (Fig. 3B,D).
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3.4 Fas, Bax (but not Bcl-2), and caspase-3 are up-regulated in Fas-induced cardiomyocyte apoptosis
As revealed by Northern blot analyses, the treatment of cardiomyocytes with FA+AD significantly enhanced the expression of the mRNA of Fas, Bax, and caspase-3 at 3.8-, 1.5-, and 2.9-fold increase, respectively (Figs. 5 and 6
). Western blotting for Fas, Bax, and caspase-3 revealed that the induction of their mRNAs was accompanied by a 2.6-, 2.2-, and 2.3-fold increase in the amount of proteins, respectively (Figs. 5 and 6). In contrast, Bcl-2 expression was not altered either at the mRNA or protein level by the treatment with FA+AD (Fig. 6). Treatments with vehicle, FA, or AD alone did not affect the expression of these factors.
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In the MKP-1-AS transfected cardiomyocytes, the Fas-induced overexpression of Fas receptor, Bax and caspase-3 was detected when treated with FA alone, while their overexpression was not noted in the JNK1-AS transfected cells even by the treatment with FA+AD (Fig. 7). This finding indicates that JNK activation is responsible for Fas-induced up-regulation of these genes.
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3.5 CAD and ICAD are constitutively expressed in cardiomyocytes
Constitutive CAD mRNA and CAD protein expression in cardiomyocytes treated with vehicle were detected in RT-PCR and Western blotting (Fig. 8). Northern and Western blots also showed ICAD expression at mRNA and protein levels in normal cardiomyocytes. The expression levels of CAD and ICAD were not affected by any of these treatments (Fig. 8).
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3.6 Caspase-3 (but not Caspase-8) and ICAD are cleaved to the active and inactive forms, respectively, in Fas-induced cardiomyocyte apoptosis
Caspase-3 is synthesized as an inactive 32-kDa protease, which is cleaved to yield two subunits of 17 and 12 kDa that tetramerize to form an active enzyme [15]. Western blot analysis demonstrated that processing of the protease was obviously detected by the appearance of the 17-kDa subunit of active caspase-3 only when treated with FA+AD (Fig. 5B). Although caspase-8 was detected in cultured cardiomyocytes, none of the treatments used in this study, including FA and AD, resulted in production of any detectable activated form of caspase-8.
ICAD is cleaved to release CAD into the nucleus to degrade DNA into internucleosomal fragments [2]. The cleaved fragment of ICAD was detectable only by treatment with FA+AD (Fig. 8B).
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4. Discussion |
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TopAbstract1. Introduction2. Methods3. Results4. DiscussionReferences |
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4.1 Classic and alternate pathways and molecular mechanisms of non-apoptosis in Fas stimulation in cardiomyocytes (Fig. 9)
Two distinct signal transduction pathways, the classic and alternate pathways, have been identified downstream of Fas in several cell lines such as HeLa, 293 and L929 cells [8,23], although they have not been examined in cardiomyocytes. In the classic pathway, the Fas-associated death domain (FADD) is recruited via the death domain of Fas receptor and catalyzes caspase-8 to become active [13]. The activated caspase-8 finally activates caspase-3 directly or through a mitochondrial cytochrome c-dependent pathway. In the present study, caspase-8 was not activated in FA+AD-induced apoptosis group and FA alone group. Therefore, the present apoptosis model is independent of the classic pathway. Moreover, Koseki et al. [10] recently reported that the expression of the apoptosis repressor with the caspase recruitment domain (ARC), an inhibitor of apoptosis, is expressed primarily and exclusively in skeletal muscle and cardiac tissue. ARC competitively inhibits binding between FADD and caspase-8. This suggests that non-activation of caspase-8 in the present study may be related to ARC which is probably independent of AD.
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The alternate pathway involves the Fas-binding protein Daxx [23]. On Fas activation, Daxx interacts with and activates a MAPK kinase kinase termed apoptosis signal-regulating kinase-1 (ASK-1), leading to the activation of JNK and p38 MAPK pathways [8,23]. JNK and p38 MAPK cascades are also activated by many kinds of stress, and they culminate in the phosphorylation and activation of transcription factors such as AP-1 and other cellular targets, and thereby are implicated to induce apoptosis [21]. Fig. 9 schematically shows hypothesized signaling pathways in Fas stimulation in cardiomyocytes. Since in this study only expression of molecules has been analyzed and not their functional involvement, the pathways downstream of Fas stimulation shown in this figure are deduced from the present findings and previous studies. The present study revealed that FA+AD induced activation of JNK, up-regulation of Bax, and activation of caspase-3 and finally DNA fragmentation and apoptotic morphologies. However, these were not induced by FA alone or AD alone. On the other hand, MKP-1, which blocks apoptosis through inhibition of JNK activation in TNF-
stimulated mesangeal cells, was up-regulated at the mRNA level by FA alone in cardiomyocytes, and the up-regulation was significantly suppressed by the additional treatment with AD. AD alone did not induce the up-regulation. Pretreatment with the AS against MKP-1 resulted in the activation of JNK and induced apoptosis in FA alone-treated cardiomyocytes, whereas the AS against JNK1 prevented apoptosis in FA+AD-treated cells. These indicate that: (1) the main pathway of FA+AD-induced apoptosis of cardiomyocytes is the alternate pathway through JNK, Bax, and caspase-3; and (2) the lack of apoptosis of cardiomyocytes by FA alone-treatment is due to inactivation of JNK via up-regulation of MKP-1. However, the mechanism of MKP-1 up-regulation by Fas stimulation is not precisely known, although the present study suggests an association with some factors that are blocked by AD.
4.2 Molecular mechanisms of up-regulation of Fas, Bax, and caspase-3 in cardiomyocytes treated with agonistic anti-Fas antibody in the presence of actinomycin D
The present study revealed up-regulation of Fas and Bax in FA+AD-treated cardiomyocytes, in which JNK activation was observed. FA alone and AD alone did not induce the above. This indicates that FA or AD itself is not a direct effector on up-regulation of Fas and Bax.
It has been reported that Fas stimulation can activate AP-1, a transcription factor, in several cell lines including cardiomyocytes [20], which is associated with activation of JNK. AP-1 is a potential enhancer of fas and bax genes [9,16]. This suggests that up-regulation of Fas and Bax in the FA+AD-treated cardiomyocytes may depend on AP-1 activation via JNK activation. That is, Fas up-regulation may be a positive feedback mechanism for progression of apoptosis.
The present study showed a precise correlation between JNK activation and caspase-3 up-regulation. Bax activates caspase-3 through releasing cytochrome c from mitochondria, but does not up-regulate caspase-3 [17]. Therefore, one of the transcription factors such as AP-1 may be associated with up-regulation of caspase-3, although no evidence was shown in the present study.
4.3 Molecular mechanism of DNA fragmentation in cardiomyocytes treated with agonistic anti-Fas antibody in the presence of actinomycin D
Recent studies have indicated that apoptotic DNA fragmentation is largely due to the action of a novel nuclease known as CAD [2]. Inactive CAD in non-apoptotic cells is a heterodimeric complex with its natural inhibitor known as ICAD. It was suggested that caspase-3-mediated ICAD cleavage occurs in the cytosol and CAD is translocated to the nucleus after it is released from the CAD/ICAD complex, where it exerts its DNase activity. In the present study, we showed for the first time constitutive expression of CAD and ICAD in cardiomyocytes. We also found that ICAD was cleaved and its fragments appeared in the apoptotic cardiomyocytes where caspase-3 was activated. Treatment with FA alone or AD alone did not result in cleavage of caspase-3 and of ICAD in cardiomyocytes. These findings suggest that DNA fragmentation in the FA+AD-treated cardiomyocytes occurs via caspase-3- and CAD-dependent pathways.
4.4 Clinical implication
Fas is constitutively expressed in cardiomyocytes [19,20]. Moreover, Fas and its mRNA are overexpressed in pathologic conditions of the heart: hypoxic cultured cardiomyocytes; salvaged myocytes in myocardial infarction; autoimmune myocarditis; dilated cardiomyopathy; volume overload; and adriamycin treatment [3]. These studies imply a possible involvement of the Fas-induced apoptosis of cardiomyocytes under pathologic conditions. However, direct evidence of apoptotic cardiomyocytes is rare in any of the above diseased conditions in vivo, in contrast to the abundant evidence of Fas overexpression. There are two possible explanations: (1) a part of the apoptotic signal transduction downstream of Fas is inhibited in cardiomyocytes, and thus cardiomyocyte apoptosis is rare; (2) apoptotic cell death does not occur continuously in cardiomyocytes but occurs intermittently (several times per year) under special conditions in diseased hearts. Since the apoptotic process occurs in a very short interval, it is very difficult to detect the cardiomyocytes undergoing apoptosis intermittently. The present findings that Fas stimulation alone does not cause apoptosis in cardiomyocytes because of inactivation of JNK via MKP-1 up-regulation confirm the concept of the former possibility. Simultaneously, the present finding that under the special conditions where MKP-1 up-regulation is prevented or MKP-1 is inactivated, cardiomyocyte apoptosis is rapidly induced by Fas stimulation also suggests the latter possibility in the diseased hearts. Further investigations are warranted.
4.5 Conclusions
Apoptotic signal transduction in the FA+AD-treated cardiomyocytes is JNK activation, Bax up-regulation, caspase-3 up-regulation, activation of CAD, and then DNA fragmentation and apoptotic morphologies including Fas up-regulation as a positive feedback mechanism. The molecular mechanism of non-apoptosis in Fas stimulation alone is inactivation of JNK via up-regulation of MKP-1.
Time for primary review 22 days.
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Acknowledgements |
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This study was supported, in part, by Grants-in-Aid for Scientific Research (No. 11670668, No. 12670704, No. 13670700 and No. 13470143) from the Ministry of Education, Science, Sports and Culture of Japan. We thank Akiko Tsujimoto for technical assistance and Daniel Mrozek for reading the manuscript.
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