Transcriptional regulation of neutral sphingomyelinase 2 in all-trans retinoic acid-treated human breast cancer cell line, MCF-7.
Transcriptional regulation of neutral sphingomyelinase 2 in all-trans retinoic acid-treated human breast cancer cell line, MCF-7.
复制标题
全反式视黄酸处理的人乳腺癌细胞系 MCF-7 中中性鞘磷脂酶 2 的转录调节。
DOI：
10.1093/jb/mvs037
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发表时间：
2012
期刊：
影响因子：
--
通讯作者：
Ito H
中科院分区：
文献类型：
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作者：
Ohba K;Oki Y;Fujita K;Kameko F;Moriyama T;Horiike Y;Morita H;Matsushita A;Iino K;Sasaki S;Nakamura H;Maekawa M.;Ito H
使用MCF-7（ATRA敏感）和MDA-MB-231（ATRA耐药）乳腺癌细胞检测全反式维甲酸（ATRA）对鞘磷脂酶表达的影响。在ATRA处理的MCF-7中观察到增加的NSMase活性、NSMase 2 mRNA和蛋白，但在ATRA处理的MDA-MB-231中未观察到。ATRA处理的MCF-7中NSMase 2 mRNA的增加主要是由于转录的增强。启动子分析显示NSMase 2重要的5′-启动子区域在−148和−42 bp之间，包含3个Sp1位点，但没有视黄酸反应元件。使用NSMase 2启动子的突变的Sp1位点、Mithramycin A（Sp抑制剂）和Sp家族过表达的实验证明了Sp家族蛋白和三个Sp1位点对于ATRA诱导的MCF-7细胞的NSMase 2转录的重要性。ATRA处理后，NSMase 2启动子区的Sp1结合量没有明显变化，但观察到了Sp1的磷酸化（活化）。有趣的是，PKCδ参与ATRA诱导的NSMase 2转录增加。ATRA诱导PKCδ磷酸化，然后激活PKCδ磷酸化的Sp1。染色质免疫沉淀（ChIP）检测显示，无论ATRA处理与否，以及ATRA诱导的组蛋白H3 5′-启动子乙酰化与否，MCF-7细胞中均有Sp1、RARα和RXRα复合物形成。因此，ATRA增强NSMase 2 mRNA表达是由于PKCδ激活引起的磷酸化Sp1的转录增加，随后是组蛋白H3乙酰化导致的染色质重塑。
Effects of all-transretinoic acid (ATRA) on sphingomyelinase expression were examined using MCF-7 (ATRA-sensitive) and MDA-MB-231 (ATRA-resistant) breast cancer cells. Increased NSMase activity, NSMase2 mRNA and protein were observed in ATRA-treated MCF-7 but not in ATRA-treated MDA-MB-231. Increased NSMase2 mRNA of ATRA-treated MCF-7 was mostly due to enhanced transcription. Promoter analysis revealed the important 5′-promoter region of NSMase2 between −148 and −42 bp containing three Sp1 sites but no retinoic acid responsive elements. Experiments using mutated Sp1 sites of the NSMase2 promoter, Mithramycin A (a Sp inhibitor) and Sp family over-expression demonstrated the importance of Sp family protein and the three Sp1 sites for ATRA-induced NSMase2 transcription of MCF-7 cells. Although no quantitative change of bound Sp1 on NSMase2 promoter region after ATRA treatment was detected, Sp1 phosphorylation (activation) by ATRA was observed. Interestingly, PKCδ was involved in ATRA-induced increased NSMase2 transcription. ATRA-induced PKCδ phosphorylation and then activated PKCδ phosphorylated Sp1. Chromatin immunoprecipitation (ChIP) assay showed Sp1, RARα and RXRα complex formation in MCF-7 cells regardless of ATRA treatment and ATRA-induced acetylated histone H3 of the 5′-promoter. Thus, NSMase2 mRNA expression enhanced by ATRA was due to increased transcription via phosphorylated Sp1 caused by PKCδ activation, followed by chromatin remodelling with histone H3 acetylation.