Hypoxia induces changes in expression of isoforms of the divalent metal transporter (DMT1) in rat pheochromocytoma (PC12) cells

Although hypoxia has been shown to increase the expression of a variety of proteins involved in iron homeostasis, including transferrin and its receptor, little is known about the effect of low oxygen on formation of isoforms of the major iron transport protein, divalent metal transporter 1, DMT1. Accordingly, we examined the effects of hypoxia on expression and subcellular distribution of the different isoforms of DMT1 in rat PC12 cells. Treatment with low oxygen modestly increased expression of protein and mRNA levels for both the +IRE and -IRE species of DMT1. In contrast, expression of the exon 1A containing species of DMT1 was greatly increased by hypoxia as indicated by Western blot and real-time RT-PCR analysis. Message levels for the 1A isoforms increased approximately 60-fold after exposure of PC12 cells to 1% oxygen for 5 h. The subcellular distribution of exon 1A isoforms of DMT1 remained consistently in the cytoplasmic milieu of the cell after hypoxic exposure, as also did the distribution of +IRE species of DMT1. The -IRE species of DMT1, however, responded to hypoxia by becoming increasingly associated with the regions adjoining the outer cellular membranes, while a portion partially colocalized with an early endosomal marker (EEA). Hypoxia also caused a significant increase in the uptake of manganese in PC12 cells. In summary, these results demonstrate that hypoxia selectively increases expression of exon 1A containing species of DMT1 with lesser increases in either the +IRE or -IRE isoforms the transporter.