Hypoxia Regulates The Divalent Metal Transporter 1 (DMT1) By Hypoxia-Inducible Factor-1 (HIF-1)

Objective: To study the effects of hypoxia on the transcription and expression of divalent metal transporter 1 (DMT1) in cells and explore the relationship between hypoxia-inducible factor-1 (HIF-1) and DMT1.Methods: Human hepatoma HepG2 cells were exposed to CoCl2 or hypoxia (1%O2). The expression of DMT1 and HIF-1αwere detected by Western Blot. The effect of hypoxia on two isoforms of DMT1 and HIF-1αsubcellular distribution were detected by immunocytochemistry. The coding sequence of HIF-1αwas constructed into expressed vector called pHIF-1α. The two different sequences from human DMT1 gene promoter were subcloned into luciferase reporter vector named pDMT1-436 and pDMT1-171. The construction containing the site-directed mutagenesis of pDMT1-171 is referred to as pDMT1-171m. Cortical astrocytes and HepG2 cells were transiently transfected with reporter constructs and expression vector construct. The luciferase activity was quantitated to identify the relationship between HIF-1αand transcription of DMT1.Results:1. HIF-1αand the two isoforms of DMT1 were increased by both hypoxia for 6 h and transiently transfected pHIF-1αin normoxia.2. In normoxia, HIF-1αexpressed little and two isoforms of DMT1 localized in both cytoplasm and nucleus uniformly in HepG2. When in hypoxia, HIF-1αexpressed predominately in nucleus, +IRE DMT1 located mainly in cytoplasm, while -IRE DMT1 changed little.3. Using the computer data base MatInspector, an extensive analysis of human DMT1 promoter revealed the presence of one putative HRE (ACGTG) at position of -327~-323. As the previously reported analysis of published HRE sequences are 5'-R(A/G)CGTG-3', we also find DMT1 promoter contains the other two putative HREs (GCGTG) at position -175~-171 and -143~-139, respectively.4. Luciferase activities from both HepG2 cells and astrocytes transfected with construct pDMT1-436 increased significiantly when exposed to hypoxia for 6h or treated with 400μM CoCl2 for 4h, demonstrating the functional HRE in human DMT1 gene promoter.5. Luciferase activities from cells transfected with construct pDMT1-436 or pDMT1-171 under hypoxia for 6h are significantly higher than that of control. No statistically differences were found between two constructs pDMT1-436 and pDMT1-171.6. No statistically differences were found among the luciferase activities from HepG2 cells transfected with pDMT1-171m in the presence or absence of hypoxia for 6h or treated with 400μM CoCl2 for 4h, suggesting that pDMT1-171 contains the functional HRE located in -327~-323 of DMT1 promoter.7. Co-transfections of pDMT1-171 with pHIF-1αobviously increased the DMT1 promoter activity. In contrast, transfection of pHIF-1αdid not affect the activity of the luciferase reporter when the DMT1 HRE was mutated.Conclusions:1. HIF-1αaccumulated and the expression of two isoforms of DMT1 increased after hypoxia and exogenous HIF-1αoverexpression in normoxic condition. The spontaneous increasing prompts that DMT1 may be regulated by HIF-1. DMT1 may be the target gene of HIF-1.2. The different changes between the distribution of +IRE DMT1 and–IRE DMT1 in HepG2 after exposed to hypoxia may be related to the different functions of two isoforms of DMT1.3. Results of luciferase expression and site-directed mutagenesis showed the region around -327~-323 (ACGTG) in DMT1 gene promoter contains a function HRE sequence.4. Significant difference in luciferase activities was observed when HepG2 was co-transfected of pDMT1-171with pHIF-1αin normoxic condition. The result prompts that HIF-1αplays a key role in DMT1 regulation in hypoxia. Hypoxia regulates DMT1 by HIF-1.