The Effects Of Hypoxic Conditions On The Expression Of The Divalent Metal Transporter 1 (DMT1)

Objective: To study the effects of hypoxic conditions on the expression of DMT1 in cultured cells and the relationship between HIF-1αand DMT1.Methods: Exposing PC12 cells and HepG2 cells to CoCl₂, the expression of +IRE DMT1 were detected by Western Blot for choosing the sensitive cell line to chemical hypoxia. Then we exposed the sensitive cell line to NaN₃, rotenone or hypoxia(1% O₂). The expression of +IRE DMT1, -IRE DMT1 and HIF-1αwere detected by Western Blot. The effect of hypoxia on the two isoforms of DMT1 and HIF-1αsubcellular distribution was detected by immunocytochemistry.Results:1. The expression of +IRE DMT1 changed little in PC12 cells which were exposed to different concentrations of CoCl₂ ( 0. 025mM,0. 05 mM,0. 125mM,0. 25 mM,0. 5mM,1 mM) for 4 hours.2. CoCl₂ could promote the expression of HIF-1αand the two isoforms of DMT1 in HepG2 cells. When HepG2 cells were exposed to 1mM CoCl₂ for 4 hours, the expression of HIF-1αreached the peak. The levels of +IRE DMT1 and - IRE DMT1 reached the highest point in HepG2 cells which were exposed to 0. 5mM CoCl₂ for 4 hours.3. Under hypoxia, HIF-1αin HepG2 cells expressed starting at 1 h hypoxia and reached the highest point at 3 to 6 h hypoxia. The level of +IRE DMT1 increased starting at 1 hour hypoxia and reached the peak at 6 h hypoxia. The expression of - IRE DMT1 increased starting at 3 h hypoxia and reached the peak at 6 h hypoxia, which lasted to 12 h.4. HIF-1αand the two isoforms of DMT1 expressed obviously in HepG2 cells which were exposed to hypoxia for 6 h. When the cells were exposed to reoxidation for 24 h, the level of +IRE DMT1 decreased while - IRE DMT1 changed little.5. When HepG2 cells were exposed to 10mM or 20mM NaN₃ for 1 h, the expression of the two isoforms of DMT1 increased significantly. The level of +IRE DMT1 in the cells also increased which were exposed to 20μM rotenone for 24 h, while -IRE DMT1 changed little. Additionally, HIF-1αcould not accumulate whenever HepG2 cells were exposed to NaN₃ or rotenone.6. In normoxia, HIF-1αexpressed little and the two isoforms of DMT1 localized in both cytoplasm and nucleus uniformly in HepG2 cells. When in hypoxia, HIF-1αexpressed significantly in the nucleus, +IRE DMT1 mainly located in cytoplasm and nuclear membrane, while the majority of - IRE DMT1 in the cytoplasm clustered closer to the cellular membrane and changed little in the nucleus.Conclusions:1. HIF-1αaccumulated and the expression of the two isoforms of DMT1 increased in HepG2 cells when they were exposed to CoCl₂ and hypoxia. The spontaneous increasing in the expression of HIF-1αand DMT1 prompt that DMT1 expression may be regulated by HIF-1. 2. When HepG2 cells were exposed to NaN₃ or rotenone, HIF-1αcould not accumulate while the expression of DMT1 increased. These phenomena demonstrate that the expression of DMT1 may be regulated through another pathway except for HIF-1.3. The different changes between the distribution of +IRE DMT1 and - IRE DMT1 in HepG2 cells after being exposed to hypoxia may be related to the different functions of +IRE DMT1 and - IRE DMT1.