Transcriptional regulation of neutral sphingomyelinase 2 gene expression of a human breast cancer cell line, MCF-7, induced by the anti-cancer drug, daunorubicin
Transcriptional regulation of neutral sphingomyelinase 2 gene expression of a human breast cancer cell line, MCF-7, induced by the anti-cancer drug, daunorubicin
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DOI：
10.1016/j.bbagrm.2009.08.006
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发表时间：
2009-11-01
影响因子：
4.7
通讯作者：
Murate, Takashi
中科院分区：
文献类型：
--
作者：
Ito, Hiromi;Murakami, Masashi;Murate, Takashi
Me2+依赖性中性SMases （NSMases）已成为应激诱导神经酰胺生产的主要候选物质。在鉴定的同工异构体中，先前的报告已经提出了NSMase2的重要性。然而，其激活机制尚未被准确报道。在此，我们分析了NSMase2基因通过抗癌药物柔红霉素（dada）表达的机制。DA增加了人乳腺癌细胞系MCF-7的细胞神经酰胺（C16、C18和C24）和NSMase活性。DA显著增加了NSMase2信息和蛋白，而NSMase1和NSMase3 mRNA的变化不大，仅观察到酸性SMase mRNA的轻度增加。NSMase2的过表达和敲低表明NSMase2在da诱导的细胞死亡中起作用。NSMase2启动子分析显示，位于第一个外显子上游- 148和- 42 bp之间的三个Sp1基序在碱性和da诱导的启动子活性中都很重要。一致地，含有三个一致的sp1基序的荧光素酶载体，而不是其突变形式，显示了da诱导的转录激活。经da处理的MCF-7显示Sp3蛋白升高。在缺乏Sp家族蛋白的SL2细胞中，Sp1和Sp3过表达均增加了NSMase启动子活性。电泳迁移率转移和ChIP分析显示，DA增加了Sp家族蛋白与三个Sp1基序的结合。(C) 2009 Elsevier B.V.版权所有
Me2+-dependent neutral SMases (NSMases) have emerged as prime candidates for stress-induced ceramide production. Among isoforms identified, previous reports have suggested the importance of NSMase2. However, its activation mechanism has not been precisely reported. Here, we analyzed the mechanism of NSMase2 gene expression by the anti-cancer drug, daunorubicin (DA). DA increased cellular ceramides (C16, C18 and C24) and NSMase activity of a human breast cancer cell line, MCF-7. DA remarkably increased the NSMase2 message and protein, whereas little change in NSMase1 and NSMase3 mRNAs and only a mild increase in acid SMase mRNA were observed. Overexpression and a knock down of NSMase2 indicated that NSMase2 played a role in DA-induced cell death. NSMase2 promoter analysis revealed that three Sp1 motifs located between - 148 and - 42 bp upstream of the first exon were important in basic as well as in DA-induced promoter activity. Consistently, luciferase vectors containing three consensus Sp1-motifs but not its mutated form showed DA-induced transcriptional activation. DA-treated MCF-7 showed increased Sp3 protein. In SL2 cells lacking Sp family proteins, both Sp1 and Sp3 overexpression increased NSMase promoter activity. Increased binding of Sp family proteins by DA to three Sp1 motifs was shown by electrophoresis mobility shift and ChIP assays. (C) 2009 Elsevier B.V. All rights reserved.