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Transcriptional regulation of the SERCA2 gene during the development of heart failure

SERCA2 基因在心力衰竭发展过程中的转录调控

基本信息

批准号：
11838001
负责人：
ARAI Masashi
金额：
$ 2.24万
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：
1999
资助国家：
日本
起止时间：
1999 至 2000
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-11838001/
关键词：
sarcoplasmic reticulum SERCA2 doxorubicin gene transcription Egr-1 ERK pressure overload Sp1 ドルソルビシン活性酸素 Egy-1 MAP kinase 心不全

项目摘要

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.

本研究探讨了阿霉素（DOX）和压力超负荷致心力衰竭过程中肌浆网Ca^<2+>-ATP酶（SERCA 2）基因转录下调的分子机制。利用培养的新生大鼠心肌细胞，我们发现H_2O_2是DOX活性的细胞内介质。SERCA 2基因5 ′侧翼区-284至-72 bp的序列具有DOX反应元件。虽然几种转录因子在SERCA 2基因的该区域具有推定的结合基序，但在DOX施用后，仅Egr-1 mRNA的表达和Egr-1蛋白与SERCA 2基因的5'调控序列的结合显著增加。我们还发现Egr-1反义寡核苷酸阻断了DOX诱导的SERCA 2 mRNA的减少，表明Egr-1是DOX诱导的心肌病中SERCA 2基因的转录抑制剂。我们观察到DOX激活三种MAP激酶，p44/42 MAP激酶，p38 MAP激酶和SAPK/JNK，但只有p44/42 MAP激酶的特异性抑制剂抑制DOX对Egr-1和SERCA 2 mRNA表达的影响。这些结果表明，活性氧的中间体，转录因子Egr-1和p44/42 MAP激酶是关键元件的SERCA 2基因的转录调控响应DOX。我们接下来确定的SERCA 2基因启动子的责任元件，其中压力过载信号转导到基因，在体内直接基因转移方法。SERCA 2基因5'端调控区中，-284 ~-72 bp的区域是压力超负荷的主要调控区域。该区域具有4个推定的Sp1转录因子结合位点。这些位点的突变分析表明，从远端的第一和第三个Sp1位点的SERCA 2基因的转录减少在压力超负荷诱导的心力衰竭中具有中心作用。