Document Type


Degree Name



Department of Cell and Developmental Biology


Oregon Health & Science University


Iron homeostasis in the body is a tightly regulated process. While iron is necessary for transport of oxygen through the body, erythropoiesis, and numerous cellular processes, too much iron is toxic to cells. Hereditary hemochromatosis (HH) is a disease that results in the accumulation of iron in the liver, heart, pancreas, and joints leading to diseases such as cirrhosis, heart failure, type II diabetes, and arthritis. HH causes the mis-regulation of body iron homeostasis and results in the absorption of too much dietary iron. Multiple tissues are involved iron homeostasis such as the intestine (which absorbs dietary iron), the liver (which senses the levels of iron in the blood and stores iron), and the macrophages of the spleen and liver (which recycle iron from senescent red blood cells) (Abstract Figure 1). All of these organs work together to ensure that enough iron (20-30mg) is available for daily erythropoiesis, while preventing the accumulation of too much iron. The majority (85-95%) of daily iron need is met by the efficient recycling of red blood cells by spleen and liver macrophages. These macrophages phagocytose senescent red blood cells, break down heme, and transport the resultant iron through the only known iron exporter, ferroportin (FPN), into the bloodstream where it is chaperoned by transferrin (Tf). The remainder of the daily iron need (1-2 mg) is met by absorption of dietary iron and needs to be very tightly regulated. Dietary iron is brought in through the apical side of enterocytes and either transported into the bloodstream on the basolateral side, when body iron is low, or stored in the enterocyte by ferritin when the body has enough iron. Iron that is stored in the enterocyte is usually lost when enterocytes are sloughed off, and excreted. The amount of FPN located on the

basolateral side of the enterocyte determines how much iron is transported into the bloodstream. FPN protein levels are regulated by hepcidin, a peptide hormone produced by the liver. Hepcidin binds to FPN and induces its internalization and degradation, making hepcidin a negative regulator of body iron uptake. Hepcidin expression is finely tuned in response to increases in iron-loaded transferrin (Tf-saturation). As the Tf-saturation increases, hepcidin is upregulated through the BMP-signaling pathway. Two proteins that are mutated in HH, HFE (the HH protein) and TFR2 (transferrin receptor 2) are hypothesized to be the Tf-saturation sensors. Mutations in either of these proteins result in loss of Tf-sensitivity, through a reduction in BMP-signaling, though how they intersect with the BMP-signaling pathway is unknown. In chapter 2 of this thesis we found that TFR2 and HFE interact with the BMP co-receptor, HJV, providing a link between BMP-signaling and Tf-sensing. HFE appears to enhance the interaction between TFR2 and HJV, providing a possible mechanism for the role of HFE in Tf-sensing. In addition, we found that all three members of the complex (TFR2, HFE, and HJV) are required for the sensing of Tf-saturation, indicating the possibility that the complex is involved in this process. Using a series of Tfr2 constructs packaged in adeno-associated virus and injected into Tfr2-deficient mice, we found the cytoplasmic domain of Tfr2 is necessary, indicating that Tf-sensing leads to modulation of BMP-signaling in the liver through intracellular domain interactions involving TFR2. These results provide an important link between Tf-saturation sensing and BMP-signaling, as well as further characterizing the functions of HJV, HFE, and TFR2 in Tf-sensing.

While the primary function of TFR2 appears to be its role in sensing Tf-saturation, work in chapter 3 of this thesis shows that TFR2 is also involved in erythropoiesis. TFR2 expression is limited to the liver and erythropoietic progenitors, and its function in these erythropoietic progenitors is unknown. Work in the lab found that spleens from TFR2 mutant mice were slightly enlarged and that this effect was not due to iron overload or a result of lack of TFR2 in the liver. In this study we show that TFR2 mutant mice show signs of stress erythropoiesis as evidenced by enlarged spleens and increased BFU-e (erythroid burst forming) and CFU-e (erythroid colony forming) assays. In addition, TFR2 mutant mice also have an elevated reticulocyte count, indicating increased erythropoiesis and consistent with stress-erythropoiesis. While a mechanism for the role of TFR2 in stress-erythropoiesis remains elusive, we provide interesting evidence that TFR2 influences erythroid maturation. In the appendix of this thesis, I further explored Tf-sensing by attempting to use a co-culture system to make a hepatoma cell line Tf-sensitive. In addition I tested the hypothesis that HFE-dependent autocrine secretion of hepcidin by macrophages would reduce FPN levels and increase intracellular iron. I also tested the effect of HFE on two isoforms of ZIP14, an iron transporter, as HFE has an effect on cellular iron loading. This work showcases the complexity of Tf-sensitivity as well as the multi-functional roles of the proteins involved.




School of Medicine



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