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Egr-1 Mediates Hypoxia-Inducible Transcription of the NDRG1 Gene through an Overlapping Egr-1/Sp1 Binding Site in the Promoter

Departments of ¹ Environmental Medicine, ² Medicine, and ³ Pharmacology, New York University School of Medicine, and New York University Cancer Institute, New York, New York

Requests for reprints: Max Costa, Nelson Institute of Environmental Medicine, New York University School of Medicine, 57 Old Forge Road, Tuxedo, NY 10987. Phone: 845-731-3515; Fax: 845-351-2118; E-mail: costam01{at}nyu.edu.

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. [Cancer Res 2007;67(19):9125–33]