|Budget Amount *help
¥3,300,000 (Direct Cost : ¥3,300,000)
Fiscal Year 1998 : ¥500,000 (Direct Cost : ¥500,000)
Fiscal Year 1997 : ¥2,800,000 (Direct Cost : ¥2,800,000)
Impaired Ca2+ handling in sarcoplasmic reticulum is a central mechanism that accounts for the cardiac dysfunction seen in doxorubicin induced-cardiomyopathy. We have recently demonstrated that mRNA expression for sarcoplasmic reticulum Ca2+-ATPase (SERCA2), the major Ca2+ transport protein in sarcoplasmic reticulum, is markedly decreased in doxorubicin induced-failing hearts.
The purpose of this study was to clarify the molecular mechanisms by which doxorubicin down-regulates SBRCA2 gene. N-acetylcysteine ; an anti-oxidant, protected SERCA2 mRNA levels from a decrease induced by doxorubicin in cultured rat cardiac myocytes, and the concentration of hydrogen peroxide was 3-fold higher than basal level, suggesting that hydrogen peroxidels an intracellular mediator of doxorubicin. Luciferase reporter assay revealed that 5' flanking sequence from -284 to -72 bp of SERCA2 gene has a doxorubicin responsible element. Of the transcription factors that have putative binding motifs in the 5' flanking -284 to -72 bp region of SERCA2 gene, the expression of Egr-1 mRNA was markedly increased after doxorubicin administration. Over-expression of Egr-1 significantly diminished the transcription of SERCA2 gene. Additionally, antisense oligonucleotides against Egr-1 rescued the doxorubicin induced-decrease of SERCA2 mRNA expression. These results suggest that Egr-1 is a transcriptional inhibitor of SERCA2 gene under doxorubicin administration. Finally, we clarified kinases which controls Egr-1 and, thus SERCA2 genes. Three MAP kinases ; p44/42 MAP kinase, p38 MAP kinase and SAPK/JNK were all activated by doxorubicin. PD98059, a specific blocker of p44/42 MAP kinase kinase, had a preventive effect on the doxorubicin induced-increase of Egr- 1 gene and the decrease of SERCA2 gene.
Our studies indicate that reactive oxygen intermediates, transcription factor Egr-1 and p44/42 MAP kinase have a pivotal role for the transcriptional regulation of SERCA2 gene in doxorubicin signaling pathway.