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How heme gets in

Intestinal transporter is regulated by hypoxia, not iron stores, says Cell study

By | September 8, 2005

The long search for an intestinal mammalian heme transporter has revealed a protein that is upregulated by hypoxia, and not iron stores, according to a study in Cell by Andrew McKie and colleagues at Kings College London.

The work adds fuel to a debate over whether heme uptake is regulated by iron levels, said Aliye Uc of the University of Iowa Children's Hospital, who did not participate in the study.

The researchers subtracted the ileum cDNA library from that of the duodenum, the primary site of intestinal heme absorption, in severely anemic mice. They honed in the protein, heme carrier protein 1 (HCP1), determining its homology to bacterial metal-tetracycline transporters and finding that its transcription is regulated by hypoxia, which is known to stimulate iron absorption.

The researchers expressed the putative transporter in xenopus oocyte cells and infected cultured HeLa cells with HCP1 adenovirus, then exposed the cell lines to radioactive heme. They found a twofold to threefold temperature-dependent increase in heme uptake with the presence of HCP1. And increasing the concentration of heme "showed that there was some saturation of uptake, suggesting a carrier-mediated event," McKie told The Scientist.

Heme analogs, but not tetracycline or free iron, were found to compete for HCP1 binding, demonstrating that the transporter recognizes the porphyrin ring enclosing the iron atom in heme. They also found that HCP1-transfected cells took up a much greater amount of a heme fluorescent analog than the control.

An HCP1-specific siRNA abolished the HCP1-induced increase in heme uptake while an HCP1-specific antibody inhibited iron absorption in intact duodenum, which Janis Abkowitz, of the University of Washington School of Medicine, called a "critical experiment" in showing that the transport plays a direct role in heme uptake.

Further experiments demonstrated that HCP1 mRNA levels are strongly induced by hypoxia but not by changes in iron stores; genes involved in non-heme iron transport, in contrast, are highly regulated by the latter. McKie said he was surprised that iron levels did not regulate HCP1 mRNA. In light of structural differences, Abkowitz said, this result suggests that different mechanisms may have evolved to deal with inorganic iron and heme.

When they localized the transporter under various conditions using electron microscopy, they found that iron status did play a role. Under iron-deficient conditions, the transporter localized to the brush-border membrane but retreated to the apical cytoplasm of the duodenal enterocytes in the presence of large iron stores, an unexplained phenomenon that McKie says has been observed in other iron transporters.

Previous work has identified heme transporters in bacteria and yeast as well as heme exporters in erythroid cells, though this is the first identification of a protein that brings heme into mammalian cells, said Abkowitz, who was not involved in the study.

McKie said the next steps will be to examine the phenotype of HCP1 knockout mice and examine transporter activity in cell-free systems. "Our kinetic studies are the first stab, really," he said. He also plans to look at the expression of HCP1 homologs in different mammalian systems.

According to Uc, the research "opens a very exciting door to the future [and] brings a lot of questions" about how heme binds to HCP1, how it is taken inside the cell, and whether there are other such transporters. "Only time will tell," she told The Scientist.

McKie said that an understanding of this transport process may inform the design of iron supplements for those affected by iron deficiency, while helping to address problems of iron overload in other populations.

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