Molecular mechanisms of non-apoptosis by Fas stimulation alone versus apoptosis with an additional actinomycin D in cultured cardiomyocytes

• Published in: Cardiovascular research 2002-09, Vol.55 (4), p.787-798
• Main Authors: AOYAMA, Takuma, TAKEMURA, Genzou, MARUYAMA, Rumi, KOSAI, Ken-Ichiro, TAKAHASHI, Tomoyuki, KODA, Masahiko, HAYAKAWA, Kenji, KAWASE, Yukinori, MINATOGUCHI, Shinya, FUJIWARA, Hisayoshi
• Format: Article
• Language: English
• Subjects: Animals; Antibodies, Monoclonal - pharmacology; Apoptosis - drug effects; Apoptosis Regulatory Proteins; Biological and medical sciences; Blotting, Northern - methods; Blotting, Western - methods; Caspase 3; Caspase 8; Caspase 9; Caspases - genetics; Cell Cycle Proteins; Cells, Cultured; Dactinomycin - pharmacology; Deoxyribonucleases - genetics; DNA, Antisense; Dual Specificity Phosphatase 1; fas Receptor - genetics; fas Receptor - immunology; fas Receptor - metabolism; Fundamental and applied biological sciences. Psychology; Gene Expression - drug effects; Genes, bcl-2; Heart; Immediate-Early Proteins - genetics; JNK Mitogen-Activated Protein Kinases; MAP Kinase Kinase 4; Mice; Mice, Inbred BALB C; Microscopy, Electron; Mitogen-Activated Protein Kinase Kinases - genetics; Mitogen-Activated Protein Kinases - genetics; Myocardium - metabolism; Myocardium - ultrastructure; p38 Mitogen-Activated Protein Kinases; Phosphoprotein Phosphatases; Protein Phosphatase 1; Protein Tyrosine Phosphatases - genetics; Proteins - genetics; Reverse Transcriptase Polymerase Chain Reaction; Transfection - methods; Vertebrates: cardiovascular system
• ISSN: 0008-6363; 1755-3245
• DOI: 10.1016/S0008-6363(02)00493-5

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. 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. 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). 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.