Iron is essential due to its presence in proteins involved in DNA synthesis, mitochondrial respiration and oxygen transport. Regulation of cellular iron content is crucial: excess cellular iron catalyzes the generation of reactive oxygen species that damage DNA and proteins, while cellular iron deficiency causes cell cycle arrest and cell death. Dysregulation of iron homeostasis caused by iron deficiency or iron excess leads to hematological, neurodegenerative and metabolic diseases in humans. Iron must therefore be maintained within a narrow range to avoid the adverse consequences of iron depletion or excess.

Maintaining iron content within this physiological range requires precise mechanisms for regulating its uptake, storage and export. In mammals, dietary non-heme Fe3+ is reduced by membrane bound ferric reductases (e.g. duodenal cytochrome B or DCYTB) before transport across the enterocyte apical membrane by divalent metal transporter-1. Cytosolic iron is either transported across the basolateral membrane into the circulation by ferroportin or sequestered in ferritin in a form unable to catalyze free radical formation. Iron export by ferroportin is dependent on oxidation to Fe3+ by membrane and soluble multicopper oxidases where it is incorporated into transferrin for delivery to tissues. When body iron stores are high, cytosolic iron is not exported into blood, and is instead sequestered into ferritin. Iron in ferritin is lost by sloughing of enterocytes into the intestinal lumen.

Mammalian intestinal iron transport increases during iron deficiency due to hypoxia-inducible factor-2? (HIF-2?) mediated expression of DMT1 and DCYTB. HIFs (HIF-1 and HIF-2) are key regulators of cellular and systemic oxygen homeostasis. HIF transcription factors consist of an oxygen-regulated ? subunit (HIF-1?, HIF-2? and a constitutively expressed ? subunit (HIF-1?, also known as aryl hydrocarbon nuclear translocator or ARNT). In the presence of iron and oxygen, HIF-? subunits are hydroxylated by iron- and oxygen-dependent prolyl hydroxylases (PHDs) and are targeted for proteasomal degradation by the von Hippel-Lindau (VHL) E3 ubiquitin ligase. During hypoxia or iron deficiency, PHDs are inactivated, allowing HIF-1? /HIF-2? to accumulate. HIF-1?/HIF-2? dimerizes with HIF-? and binds to HREs in target genes to increase transcription. HIF regulates genes in diverse pathways including erythropoiesis, iron homeostasis, glucose metabolism, angiogenesis and cell survival.

Oxygen and iron homeostasis pathways are conserved in Caenorhabditis elegans. The HIF-1 pathway in C. elegans consists of hif-1, aha-1, vhl-1 and egl-9, which are orthologous to genes encoding mammalian HIF-1?, HIF-1?, VHL and PHD. elegant express a single hif gene, which encodes a protein homologous to vertebrate HIF-1? and HIF-2?. elegans HIF-1 regulates target genes involved in metabolism, extracellular remodeling, nervous system development, oxygen-dependent behavior and modulation of life span. hif-1 mutant animals display increased embryonic and larval lethality in oxygen concentrations less than 1%, demonstrating the importance of HIF-1 for survival during hypoxia.

C. elegans express genes homologous to ferritin (ftn-1 and ftn-2), DMT1 (smf-1, smf-2 and smf-3) and ferroportin (fpn-1.1, fpn-1.2 and fpn-1.3). Vertebrate ferritin is a mixture of 24 light-(L) and heavy-(H) subunits that form a shell that can accommodate up to 4500 iron atoms. The H-subunits exhibit ferroxidase activity and facilitate the oxidation of iron, whereas the L-subunits function with the H-subunits in iron nucleation.

C. elegans FTN-1 and FTN-2 display greater homology to the human H-subunit (55% and 60%) than to the L-subunit (46% and 50%), and notably both proteins contain ferroxidase active-site residues. ftn-1 and ftn-2 genes are transcriptionally repressed during iron deficiency, which is dependent on an iron-dependent enhancer (IDE) located in the ftn-1 and ftn-2 promoters. The IDE contains two GATA binding sites for the intestinal specific ELT2 transcription factor that regulates basal ftn-1 and ftn-2 transcription, but the mechanism regulating iron-dependent transcriptional repression is unknown. Unlike ftn-1 and ftn-2, vertebrate ferritin-H and -L subunit mRNAs are translationally repressed by iron-regulatory proteins 1 and 2 (IRP1 and IRP2) during iron deficiency. SMF-1, SMF-2 and SMF-3 display 55–58% amino acid identity with mammalian DMT1 and are involved in Mn2+ uptake and sensing, but the role of these transporters in iron uptake is not well understood. The function of ferroportin homologs FPN1.1, FPN-1.2 and FPN-1.3 in iron homeostasis has not been reported.

Here, we show that HIF-1 activates smf-3 transcription and inhibits ftn-1 and ftn-2 transcription during iron deficiency. Transcriptional activation of smf-3 and repression of ftn-1 and ftn-2 is dependent on IDEs in their promoters that are similar but not identical. These studies show that HIF-1 is a key regulator of intestinal iron uptake and storage during iron deficiency in C. elegans.p>