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

肌浆网钙离子转运受损是阿霉素所致心肌病心脏功能障碍的主要机制。最近，我们发现肌浆网中主要的钙转运蛋白肌浆网钙-ATPase(SERCA2)的mRNA表达在阿霉素诱导的心力衰竭中显著降低，本研究旨在阐明阿霉素下调SBRCA2基因的分子机制。抗氧化剂N-乙酰半胱氨酸可保护阿霉素引起的大鼠心肌细胞SERCA2基因表达水平的下降，过氧化氢浓度是基础水平的3倍，提示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，SERCA2基因-284~-72bp的5‘侧翼序列含有阿霉素敏感元件。在SERCA2基因5‘侧翼-284~-72bp区域具有可能结合基序的转录因子中，给予阿霉素后Egr-1mRNA的表达显著增加。Egr-1的过表达显著抑制了SERCA2基因的转录。此外，针对Egr-1的反义寡核苷酸可挽救阿霉素诱导的SERCA2基因表达的降低。这些结果表明，Egr-1是阿霉素作用下SERCA2基因的转录抑制因子。最后，我们阐明了控制Egr-1和SERCA2基因的激酶。阿霉素可激活p44/42、p38和SAPK/JNK三种MAP激酶。P44/42 MAP激酶的特异性阻断剂PD98059对阿霉素诱导的Egr-1基因的上调和SERCA2基因的降低具有预防作用，我们的研究表明，活性氧中间体、转录因子Egr-1和p44/42 MAP激酶在阿霉素信号通路中对SERCA2基因的转录调控起着关键作用。

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.