|Budget Amount *help
¥4,000,000 (Direct Cost: ¥4,000,000)
Fiscal Year 2002: ¥1,600,000 (Direct Cost: ¥1,600,000)
Fiscal Year 2001: ¥2,400,000 (Direct Cost: ¥2,400,000)
Cultured neonatal mouse cardiomyocytes were treated with agonistic anti-Fas antibody (FA), AD, or both (FA + AD). In this system, apoptotic signals related with Fas-induced apoptotic pathways were examined by RT-PCR and immunoblottings. In addition, antisense oligonucleotide (AS) studies were performed. 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 mutagen-activated protein kinase phosphatase-1 (MKP-1), an in activator 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 extra cellular signal-regulated kinase (ERK). In conclusion, 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.