Iron overload diseases are among the most common genetic disorders worldwide. This proposal analyzes the molecular pathways that lead to iron overload diseases, including hereditary hemochromatosis. Recent discoveries created new opportunities for fundamental molecular understanding of iron metabolism and the diseases of iron overload. The peptide hormone hepcidin emerged as the principal regulator of systemic iron homeostasis. Hepcidin synthesis is induced by inflammation and by iron loading in vivo, and is suppressed by hypoxia and anemia. In turn, hepcidin inhibits the intestinal absorption of dietary iron, as well as iron recycling by macrophages and iron release from hepatic stores. Hepcidin deficiency and the resulting dysregulation of iron absorption are the common pathogenetic features of most hereditary hemochromatoses, except for those caused by mutations in the hepcidin target, ferroportin. The molecular pathways that sense iron and regulate hepcidin synthesis in response to iron loading are not known. It is also not clear how the specific genes mutated in hemochromatosis cause hepcidin deficiency. We reason that the hemochromatosis genes HFE, transferrin receptor 2 and hemojuvelin, whose homozygous disruption causes partial or complete deficiency of hepcidin, encode proteins that regulate hepcidin production. Of these genes, hemojuvelin appears to be the "closest" to hepcidin because its homozygous disruption causes the absence or near absence of hepcidin. Moreover, the disruption of hemojuvelin is the main cause of juvenile hemochromatosis, and phenotypically completely mimics homozygous disruption of the hepcidin gene itself. Furthermore, hemojuvelin belongs to a family of receptor ligands, repulsive guidance molecules (RGM), indicating that it may function in a signal transduction pathway. By focusing on hemojuvelin and identifying its partners and function, this R21 proposal will lay the groundwork for understanding how iron regulates hepcidin production. Specifically, we will: 1. Prepare and characterize human hemojuvelin and generate anti-hemojuvelin antibodies. 2. Identify the hemojuvelin receptor(s). 3. Analyze the regulation of hemojuvelin synthesis and processing by iron and inflammation 4. Identify the biological effects of hemojuvelin in vivo. The results of this work will form the foundation of a comprehensive R01 proposal to elucidate how iron load is sensed and how it regulates hepcidin production, processes that constitute the afferent arc of systemic regulation of iron absorption and transport. [unreadable] [unreadable]