ATRA inhibits ceramide kinase transcription in a human neuroblastoma cell line, SH-SY5Y cells: the role of COUP-TFI
ATRA inhibits ceramide kinase transcription in a human neuroblastoma cell line, SH-SY5Y cells: the role of COUP-TFI
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DOI：
10.1111/j.1471-4159.2009.06486.x
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发表时间：
2010-01-01
影响因子：
4.7
通讯作者：
Murate, Takashi
中科院分区：
文献类型：
--
作者：
Murakami, Masashi;Ito, Hiromi;Murate, Takashi
神经酰胺是鞘磷脂代谢的中心脂类。神经酰胺激酶(Cerk)及其产物1-磷酸神经酰胺参与了多种细胞功能。然而，CERK基因表达的调控机制尚不清楚。在这里，我们检测了全反式维甲酸(ATRA)诱导人神经母细胞瘤细胞系SH-SY5Y分化过程中Cerk mRNA的水平。全反式维甲酸可降低CERK的mRNA和蛋白水平。CERK的过度表达和小干扰RNA(SiRNA)表明，CERK对ATRA诱导的神经元分化和细胞生长停滞具有抑制作用。全反式维甲酸抑制CERK基因5‘端启动子的转录活性。截短和突变研究表明，全反式维甲酸反应区主要位于-40bp至第一外显子之间的串联维甲酸反应元件(REARE)上。凝胶迁移率改变分析表明，ATRA产生两条延缓条带，分别被抗鸡卵蛋白上游转录因子I(COUP-TFI)、RARα和RXRα抗体消除。DNA下拉实验证实，这些转录因子与REARE的结合增加。RAR、RXR和COUP-TFI的瞬时表达和siRNA转染表明COUP-TFI抑制了Cerk基因的表达。染色质免疫沉淀实验显示共抑制因子和三种转录因子的募集。这些结果表明COUP-TFI是CERK基因转录的ATRA反应性抑制转录因子。
Ceramide is the central lipid in the sphingolipid metabolism. Ceramide kinase (CERK) and its product, ceramide 1-phosphate, have been implicated in various cellular functions. However, the regulatory mechanism of CERK gene expression remains to be determined. Here, we examined CERK mRNA level during all-trans retinoic acid (ATRA)-induced differentiation of a human neuroblastoma cell line, SH-SY5Y. ATRA reduced CERK mRNA and protein levels. Over-expression and small interfering RNA (siRNA) of CERK revealed that CERK is inhibitory against ATRA-induced neuronal differentiation and cell growth arrest. ATRA inhibited the transcriptional activity of 5'-promoter of CERK. Truncation and mutation study suggests that ATRA-responsible region was mainly located in the tandem retinoic acid responsive elements (RARE) between -40 bp and the first exon. The electrophoresis mobility shift assay revealed that ATRA produced two retarded bands, which were erased by antibody against chicken ovalbumin upstream promoter transcription factor I (COUP-TFI), RAR alpha, and RXR alpha, respectively. DNA pull-down assay confirmed increased binding of these transcription factors to RARE. Transient expression of RAR, RXR, and COUP-TFI and siRNA transfection of these genes revealed that COUP-TFI inhibited CERK mRNA. Furthermore, chromatin immunoprecipitation assay showed the recruitment of co-repressors as well as three transcription factors. These results suggest that COUP-TFI was the ATRA-responsive suppressive transcription factor of CERK gene transcription.