Hepcidin is a key iron regulatory hormone that controls expression of the iron exporter ferroportin to increase the iron supply when needed to support erythropoiesis and other essential functions, but to prevent the toxicity of iron excess. Abnormally low hepcidin expression leads to the iron overload disorder hereditary hemochromatosis and contributes to iron loading anemias such as b-thalassemia. Excess hepcidin contributes to iron restricted erythropoiesis and anemia in a number of chronic diseases. A key unanswered question is how the liver senses iron levels in the body to appropriately coordinate hepcidin expression. We previously discovered that the bone morphogenetic protein (BMP)6-SMAD signaling pathway is a central regulator of hepcidin transcription in response to iron. Moreover, modulators of the BMP6-SMAD pathway regulate hepcidin expression to treat hemochromatosis and anemia of chronic disease in animal models. These studies have already yielded important insights into the pathophysiology of hemochromatosis and have identified the BMP6-SMAD pathway as a viable therapeutic target for iron disorders. However, it remains largely unknown how iron is sensed by the liver to regulate BMP6-SMAD signaling and thereby hepcidin production. In the last funding period, we discovered that liver endothelial cells are a key source for BMP6 in the regulation of hepcidin production. We also established a primary liver endothelial cell culture model and demonstrated that BMP6 transcription in liver endothelial cells is governed by intracellular iron content. Finally, we used this cell culture model in conjunction with quantitative proteomics and RNA-seq screens to identify iron transporters and transcription factor pathways that we hypothesize play a key role in mediating BMP6 production in response to iron to control hepcidin expression and systemic iron homeostasis. In Specific Aim I, we will use primary liver endothelial cultures and genetic mouse models to establish the role of specific iron transporters in controlling liver endothelial cell iron content and iron-regulated pathways to govern BMP6 expression. In Specific Aim II, we will use primary liver endothelial cell cultures, chromatin immunoprecipitation, reporter assays, pharmacologic approaches, and endothelial conditional knockout mouse models to determine the role of candidate transcription factor pathways and their mechanism of activation in controlling BMP6 production in response to iron. The long-term goals of this project are to understand how the BMP signaling pathway is modulated by different signals to regulate hepcidin expression and systemic iron balance, to gain insights into the physiology and pathophysiology of iron homeostasis in health and disease, and ultimately to develop new therapeutic strategies for treating disorders of iron metabolism.