Molecular mechanisms of non-apoptosis by Fas stimulation alone versus apoptosis with an additional actinomycin D in cultured cardiomyocytes.
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.
Aoyama
Takuma
T
Second Department of Internal Medicine, Gifu University School of Medicine, 40 Tsukasa-Machi, Gifu 500-8705, Japan.
Takemura
Genzou
G
Maruyama
Rumi
R
Kosai
Ken-ichiro
K
Takahashi
Tomoyuki
T
Koda
Masahiko
M
Hayakawa
Kenji
K
Kawase
Yukinori
Y
Minatoguchi
Shinya
S
Fujiwara
Hisayoshi
H
D000818:Animals; D000911:Antibodies, Monoclonal / Q000494:pharmacology; D017209:Apoptosis / Q000187:drug effects*; D051017:Apoptosis Regulatory Proteins; D015152:Blotting, Northern / Q000379:methods; D015153:Blotting, Western / Q000379:methods; D053148:Caspase 3; D053181:Caspase 8; D053453:Caspase 9; D020169:Caspases / Q000235:genetics; D018797:Cell Cycle Proteins; D002478:Cells, Cultured; D016373:DNA, Antisense; D003609:Dactinomycin / Q000494:pharmacology*; D003851:Deoxyribonucleases / Q000235:genetics; D054638:Dual Specificity Phosphatase 1; D015870:Gene Expression / Q000187:drug effects; D019254:Genes, bcl-2; D017874:Immediate-Early Proteins / Q000235:genetics; D048031:JNK Mitogen-Activated Protein Kinases; D048670:MAP Kinase Kinase 4; D051379:Mice; D008807:Mice, Inbred BALB C; D008854:Microscopy, Electron; D020929:Mitogen-Activated Protein Kinase Kinases / Q000235:genetics; D020928:Mitogen-Activated Protein Kinases / Q000235:genetics; D009206:Myocardium / Q000378:metabolism* / Q000648:ultrastructure; D010749:Phosphoprotein Phosphatases; D054645:Protein Phosphatase 1; D017027:Protein Tyrosine Phosphatases / Q000235:genetics; D011506:Proteins / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014162:Transfection / Q000379:methods; D019014:fas Receptor / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D048051:p38 Mitogen-Activated Protein Kinases
D000911:Antibodies, Monoclonal; D051017:Apoptosis Regulatory Proteins; D018797:Cell Cycle Proteins; D016373:DNA, Antisense; D017874:Immediate-Early Proteins; D011506:Proteins; C109664:caspase-activated DNase inhibitor; D019014:fas Receptor; D003609:Dactinomycin; D048031:JNK Mitogen-Activated Protein Kinases; D020928:Mitogen-Activated Protein Kinases; D048051:p38 Mitogen-Activated Protein Kinases; D048670:MAP Kinase Kinase 4; D020929:Mitogen-Activated Protein Kinase Kinases; D003851:Deoxyribonucleases; C109663:caspase-activated deoxyribonuclease; D010749:Phosphoprotein Phosphatases; D054645:Protein Phosphatase 1; D054638:Dual Specificity Phosphatase 1; C517020:Dusp1 protein, mouse; D017027:Protein Tyrosine Phosphatases; C505409:Casp3 protein, mouse; C505526:Casp8 protein, mouse; C506434:Casp9 protein, mouse; D053148:Caspase 3; D053181:Caspase 8; D053453:Caspase 9; D020169:Caspases