Divalent Metal Transporter 1 is a Hypoxia-Inducible Gene

Our recent study revealed a high correlation between the expression of hypoxia-inducible factor-1 (HIF-1) alpha and divalent metal transporter 1 (DMT1) in HepG2 cells treated with chemical or physical hypoxia. We therefore speculated that DMT1 might be one of the target genes of HIF-1. Here, we characterized the DMT1 exon 1B promoter region and identified a functional hypoxia response element (HRE, 5'-TCAGTACCTAACGTGGCGCCACGGC-3') harboring a binding site for HIF-1. We demonstrated that hypoxia-dependent activation of a luciferase reporter gene in transfected HepG2 cells is mediated by a fragment of human DMT1 exon 1B promoter containing the putative HRE sequence. We also showed that the HIF-1 binding site (HBS) is in DMT1 exon 1B promoter with the core sequence of HRE (5'-ACGTG-3') at -327 to -323 relative to the transcription start site of the human DMT1 exon 1B gene. The mutation of this sequence prevented stimulation of luciferase activity. Electrophoretic mobility shift assays revealed that the HRE sequence found in the DMT1 gene promoter was bound by HIF-1. In addition, we provide evidence that hypoxia could significantly increase ferrous uptake, while the silencing of total DMT1 by RNA interference down-regulates DMT1 expression and ferrous uptake in HepG2 cells. We conclude that DMT1 is a hypoxia-inducible gene. J. Cell. Physiol. 226: 1596-1603, 2011. (C) 2010 Wiley-Liss, Inc.