| Budget Amount *help |
¥3,500,000 (Direct Cost: ¥3,500,000)
Fiscal Year 2000: ¥900,000 (Direct Cost: ¥900,000)
Fiscal Year 1999: ¥2,600,000 (Direct Cost: ¥2,600,000)
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| Research Abstract |
In this research, we have dissected the molecular mechanisms which down-regulates sarcoplasmic reticulum Ca^<2+>-ATPase (SERCA2) gene transcription during the development of heart failure induced by doxorubicin (DOX) and pressure overload. Using cultured rat neonatal cardiac myocytes, we found that H2O2 is an intracellular mediator of DOX activity. The sequence from -284 to -72 bp in the 5' flanking region of the SERCA2 gene has a DOX-responsive element. While several transcription factors have putative binding motifs in this region of the SERCA2 gene, only the expression of Egr-1 mRNA and the binding of Egr-1 protein to the 5' regulatory sequence of SERCA2 gene increased markedly after DOX administration. We also found that Egr-1 antisense oligonucleotides blocked the DOX-induced reduction in SERCA2 mRNA, suggesting that Egr-1 is a transcriptional inhibitor of the SERCA2 gene in DOX-induced cardiomyopathy. We observed activation of three MAP kinases, p44/42 MAP kinase, p38 MAP kinase and SAPK/JNK, by DOX, but only a specific inhibitor of the p44/42 MAP kinase kinase, suppressed the effects of DOX on Egr-1 and SERCA2 mRNA expression. These findings indicate that reactive oxygen intermediates, the transcription factor Egr-1 and p44/42 MAP kinase are critical elements in the transcriptional regulation of the SERCA2 gene in response to DOX.We next determined the responsible elements of SERCA2 gene promoter, in which pressure overload signal is transduced to the gene, using in vivo direct gene transfer method. Of the 5' regulatory region of SERCA2 gene, the region spanning from -284 to -72 bp is responsible for the pressure overload. This region has 4 putative Sp1 transcription factor binding sites. Mutagenesis analysis of these sites revealed that first and third Sp1 sites from distal end have a central role for the reduced transcription of SERCA2 gene in pressure overload-induced heart failure.
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