ABSTRACT Dysregulation of intestinal iron absorption is a primary feature in iron overload disorders. Hypoxia-inducible factor 2α (HIF2α) is a critical regulator of iron absorption during increased systemic iron demands (i.e. iron deficiency, erythropoiesis, and pregnancy). HIF2α regulates expression of the apical iron transport machinery, divalent metal transporter 1 (DMT-1) and duodenal cytochrome b (Dcytb), and regulates the basolateral iron exporter ferroportin independent of hepcidin, the master systemic regulator of iron homeostasis. Disrupting intestinal HIF2α decreases tissue iron accumulation in iron overload disorders, such as β-thalassemia and heredity hemochromatosis. Moreover, in β-thalassemia, disruption of intestinal HIF2α also improves anemia. This has laid the foundation for HIF2α-based therapeutics for β-thalassemia and heredity hemochromatosis, an area actively being researched by Peloton Therapeutics. Although our results demonstrate a central role for HIF2α in intestinal iron absorption, the underlying mechanisms behind its overlapping and distinct roles in iron deficiency, β-thalassemia, and heredity hemochromatosis are still unclear. We hypothesize that a decrease in systemic hepcidin triggers HIF2α activation in intestinal epithelia, leading to an iron-absorptive response, which is critical for tissue iron accumulation in iron overload disorders. Our long-term goals are to improve existing HIF2α-based therapies and identify novel HIF2α-based therapies in iron-related disorders. The major goal of this proposal is to assess the precise mechanisms by which HIF2α selectively regulates iron absorption and determine if inhibition of HIF2α signaling and downstream mediators can be used to restrict tissue iron overload. We will pursue our objectives through three interconnected Specific Aims. Aim 1 will identify mechanisms leading to rapid activation of HIF2α in hereditary hemochromatosis. Our data suggest a crosstalk between the systemic iron regulator, hepcidin, and intestinal HIF2α. This concept will be tested in novel mouse models that allow us to acutely and temporally regulate the hepcidin-ferroportin axis. Aim 2 will characterize precise mechanisms leading to an iron-absorptive HIFα response. We have identified mothers against decapentaplegic homolog 3 (SMAD3) as a novel factor that is essential for expression of iron- absorptive (but not glycolytic, angiogenic, or inflammatory) HIF2α target genes. We will elucidate the specific role and underlying mechanisms behind SMAD3 regulation of HIF2α signaling. Aim 3 will assess the requirement for HIF2α-induced intestinal ferritinophagy in systemic iron homeostasis and iron overload disorders. Nuclear coactivator-4 (NCOA4), the major regulator of ferritinophagy, is directly regulated by HIF2α and is highly induced in iron deficiency, β-thalassemia, and heredity hemochromatosis. We will examine the role that autophagic ferritin turnover plays in iron absorption using novel mouse mod...