Arsenite induces a cell stress-response gene, RTP801, through reactive oxygen species and transcription factors Elk-1 and CCAAT/enhancer-binding protein
Arsenite induces a cell stress-response gene, RTP801, through reactive oxygen species and transcription factors Elk-1 and CCAAT/enhancer-binding protein
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DOI：
10.1042/bj20050553
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发表时间：
2005-11-15
影响因子：
4.1
通讯作者：
Chen, Y
中科院分区：
文献类型：
--
作者：
Lin, L;Stringfield, TM;Chen, Y
RTP801是一个新发现的应激反应基因，由缺氧和其他细胞应激信号诱导。砷是一种与人类致癌有关的重金属。在这里，我们研究了砷诱导RTP801转录的机制。在HaCaT人角质形成细胞中，亚砷酸盐能够诱导RTP801 mRNA水平的快速上升。相应地，亚砷酸盐处理能够刺激2.5 kb的人RTP801启动子。这种刺激作用被抑制的超氧化物歧化酶或谷胱甘肽过氧化物酶的共表达，并废除了N-乙酰半胱氨酸，这意味着活性氧（活性氧）参与了RTP801基因的转录调控。对该启动子的一系列缺失研究显示，在启动子的-1057和-981 bp之间存在一个关键的砷响应区域。点突变的推定埃尔克-1网站和C/EBP（CCAAT/增强子结合蛋白）网站在这一地区能够减少砷的刺激作用，表明埃尔克-1和C/EBP参与转录调控的RTP801基因的亚砷酸盐。此外，凝胶迁移率变化试验表明，亚砷酸盐能够安装的快速形成的蛋白质复合物，结合砷的响应区域以及C/EBP-含有序列。砷对RTP801转录的刺激部分是由ERK（细胞外信号调节激酶）途径介导的，因为RTP801的作用被选择性ERK抑制剂抑制。此外，Elk-1和C/EBP β的过表达能够提高启动子活性。因此，这些研究表明，RTP801是砷在人角质形成细胞中的转录靶点，并且砷和ROS的产生与Elk-1和C/EBP在转录控制中相关。
RTP801 is a newly discovered stress-response gene that is induced by hypoxia and other cell stress signals. Arsenic is a heavy metal that is linked to carcinogenesis in humans. Here, we investigated the mechanism by which arsenic induces RTP801 transcription. In HaCaT human keratinocytes, arsenite was able to induce a rapid rise in the RTP801 mRNA level. Correspondingly, arsenite treatment was capable of stimulating a 2.5 kb human RTP801 promoter. Such a stimulatory effect was inhibited by co-expression of superoxide dismutase or glutathione peroxidase, and was abrogated by N-acetylcysteine, implying that ROS (reactive oxygen species) were involved in transcriptional regulation of the RTP801 gene. A series of deletion studies with the promoter revealed a critical arsenic-responsive region between - 1057 and - 981 bp of the promoter. Point mutations of the putative Elk-1 site and the C/EBP (CCAAT/enhancer-binding protein) site within this region were able to reduce the stimulatory effect of arsenite, indicating that Elk-1 and C/EBP are involved in transcriptional regulation of the RTP801 gene by arsenite. Furthermore, a gel mobility-shift assay demonstrated that arsenite was able to mount the rapid formation of a protein complex that bound the arsenic-responsive region as well as the C/EBP-containing sequence. The arsenite stimulation on RTP801 transcription was partly mediated by the ERK (extracellular-signal-regulated kinase) pathway, since the effect of RTP801 was inhibited by a selective ERK inhibitor. In addition, overexpression of Elk-1 and C/EBP beta was able to elevate the promoter activity. Therefore these studies indicate that RTP801 is a transcriptional target of arsenic in human keratinocytes, and that arsenic and ROS production are linked to Elk-1 and C/EBP in the transcriptional control.