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Analyses of transcriptional regulation and signal transduction of sarcoplasmic reticulum Ca^<2+>

肌浆网Ca^<2>转录调控及信号转导分析

基本信息

批准号：
09670692
负责人：
ARAI Masashi
金额：
$ 2.11万
依托单位：
Gunma University School of Medicine
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
1997
资助国家：
日本
起止时间：
1997 至 1998
项目状态：
已结题

项目摘要

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.

肌浆网中Ca 2+处理受损是多柔比星诱导的心肌病中观察到的心功能不全的中心机制。本研究旨在阐明阿霉素下调心肌肌浆网Ca ~（2+）-ATP酶（sarcoplasmic reticulum Ca ~（2+）-ATPase，SERCA_2）基因表达的分子机制。乙酰半胱氨酸;在培养的大鼠心肌细胞中，一种抗氧化剂，保护SERCA 2 mRNA水平免受阿霉素诱导的降低，并且过氧化氢的浓度比基础水平高3倍，表明过氧化氢是阿霉素的细胞内介质。荧光素酶报告基因分析表明SERCA 2基因5'端-284 ~-72 bp的侧翼序列具有阿霉素的反应元件。在SERCA 2基因5'端-284 ~-72 bp区域具有结合基序的转录因子中，阿霉素诱导Egr-1 mRNA表达显著增加。Egr-1的过表达显著降低了SERCA 2基因的转录。此外，针对Egr-1的反义寡核苷酸挽救了阿霉素诱导的SERCA 2 mRNA表达的降低。这些结果表明，Egr-1是阿霉素给药下SERCA 2基因的转录抑制剂。最后，我们阐明了控制Egr-1和SERCA 2基因的激酶。三种MAP激酶：p44/42 MAP激酶、p38 MAP激酶和SAPK/JNK均被阿霉素激活。p44/42 MAP激酶的特异性阻断剂PD 98059对阿霉素诱导的Egr- 1基因表达的增加和SERCA 2基因表达的减少具有预防作用，提示活性氧中间产物、转录因子Egr-1和p44/42 MAP激酶在阿霉素信号通路中对SERCA 2基因的转录调控起关键作用。