Arsenite induces a cell stress-response gene, RTP801, through reactive oxygen species and transcription factors Elk-1 and CCAAT/enhancer-binding protein.
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/EBPbeta 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.
Lin
Lin
L
Department of Medical and Molecular Genetics, Walther Oncology Center, Indiana University School of Medicine and the Walther Cancer Institute, Indianapolis, IN 46202, USA.
Stringfield
Teresa M
TM
Shi
Xianglin
X
Chen
Yan
Y
D017209:Apoptosis; D018053:Arsenites / Q000494:pharmacology*; D001483:Base Sequence; D022782:CCAAT-Enhancer-Binding Protein-beta / Q000378:metabolism*; D002460:Cell Line; D005786:Gene Expression Regulation / Q000187:drug effects*; D006801:Humans; D008969:Molecular Sequence Data; D011401:Promoter Regions, Genetic; D012333:RNA, Messenger / Q000378:metabolism; D017382:Reactive Oxygen Species / Q000378:metabolism*; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism*; D050807:ets-Domain Protein Elk-1 / Q000378:metabolism*
D018053:Arsenites; D022782:CCAAT-Enhancer-Binding Protein-beta; C469991:DDIT4 protein, human; D012333:RNA, Messenger; D017382:Reactive Oxygen Species; D014157:Transcription Factors; D050807:ets-Domain Protein Elk-1; C015001:arsenite