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Title: Egr-1 mediates hypoxia-inducible transcription of the NDRG1 gene through an overlapping Egr-1/Sp1 binding site in the promoter.

Authors: Zhang, Ping; Tchou-Wong, Kam-Meng; Costa, Max

Published In Cancer Res, (2007 Oct 1)

Abstract: N-myc down-regulated gene 1 (NDRG1/Cap43) is inducible by a variety of environmental stressors, including hypoxia. The present study identified a cis-acting element mediating the transactivation of the NDRG1 gene in murine RAW264.7 macrophage cells treated with hypoxia or deferoxamine, an iron chelator mimicking hypoxia. Through a series of deletions of the promoter of NDRG1 luciferase constructs, a minimal cis-acting element conferring inducibility by hypoxia and deferoxamine was localized to an early growth response 1 (Egr-1) and Sp1 overlapping binding site. Electrophoretic mobility shift assay, antibody supershift assay, and mutations of the Egr-1 binding site confirmed the specific binding of Egr-1 protein to this Egr-1/Sp1 motif. In addition, hypoxia increased the level of Egr-1 protein that correlated with induction of NDRG1 expression at both RNA and protein levels. Transient transfection of the Egr-1 gene into HeLa cells also resulted in up-regulation of the NDRG1 mRNA. The role of Egr-1 was further verified by mutations in the Egr-1 binding site, which reduced promoter inducibility by hypoxia and deferoxamine. Furthermore, the induction of NDRG1 expression by hypoxia and deferoxamine was diminished by RNA interference knockdown of Egr-1 gene expression and in Egr-1-/- mouse embryonic fibroblasts (MEF) compared with Egr-1+/- MEFs. These results showed for the first time that Egr-1 regulates NDRG1 transcription through an overlapping Egr-1/Sp1 binding site that acts as a major site of positive regulation of the NDRG1 promoter by hypoxia signaling.

PubMed ID: 17909017 Exiting the NIEHS site

MeSH Terms: Animals; Binding Sites; Cell Cycle Proteins/biosynthesis; Cell Cycle Proteins/genetics*; Cell Hypoxia/genetics; Cell Line, Tumor; Deferoxamine/pharmacology; Early Growth Response Protein 1/genetics*; Early Growth Response Protein 1/metabolism; Gene Expression Regulation, Neoplastic; Hela Cells; Humans; Intracellular Signaling Peptides and Proteins/genetics*; Macrophages/metabolism; Macrophages/physiology; Mice; Mutagenesis, Site-Directed; Okadaic Acid/pharmacology; Promoter Regions (Genetics); Sp1 Transcription Factor/genetics*; Sp1 Transcription Factor/metabolism; Transcription, Genetic*; Transfection; Up-Regulation

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