Abstract
Liver sinusoidal endothelial cell–derived bone morphogenetic protein 6 (BMP6) and the BMP6–small mothers against decapentaplegic homolog (SMAD) signaling pathway are essential for the expression of hepcidin, the secretion of which is considered the systemic master switch of iron homeostasis. However, there are continued controversies related to the strong and direct suppressive effect of iron on hepatocellular hepcidin in vitro in contrast to in vivo conditions. Here, we directly studied the crosstalk between endothelial cells (ECs) and hepatocytes using in vitro coculture models that mimic hepcidin signaling in vivo. Huh7 cells were directly cocultured with ECs, and EC conditioned media (CM) were also used to culture Huh7 cells and primary mouse hepatocytes. To explore the reactions of ECs to surrounding iron, they were grown in the presence of ferric ammonium citrate and heme, two iron-containing molecules. We found that both direct coculture with ECs and EC-CM significantly increased hepcidin expression in Huh7 cells. The upstream SMAD pathway, including phosphorylated SMAD1/5/8, SMAD1, and inhibitor of DNA binding 1, was induced by EC-CM, promoting hepcidin expression. Efficient blockage of this EC-mediated hepcidin upregulation by an inhibitor of the BMP6 receptor ALK receptor tyrosine kinase 2/3 or BMP6 siRNA identified BMP6 as a major hepcidin regulator in this coculture system, which highly fits the model of hepcidin regulation by iron in vivo. In addition, EC-derived BMP6 and hepcidin were highly sensitive to levels of not only ferric iron but also heme as low as 500 nM. We here establish a hepatocyte–endothelial coculture system to fully recapitulate iron regulation by hepcidin using EC-derived BMP6.
Highlights
Iron is essential to the metabolism of all cells and highly toxic in the light of Fenton chemistry [1]
endothelial cells (EC) strongly enhance hepcidin in liver cells We used two established EC lines to study the crosstalk between ECs and Huh7 cells in detail
Human Umbilical Vein Endothelial Cells (HUVEC) are isolated from human umbilical veins and they are widely used as a normal endothelial cell line [41,42]
Summary
Iron is essential to the metabolism of all cells and highly toxic in the light of Fenton chemistry [1]. Hepcidin suppression has been recognized as major mechanisms of chronic iron accumulation in most hereditary iron overload diseases such as hemochromatosis [17], hepcidin is seemingly paradoxically and profoundly suppressed in hemolytic diseases such as thalassemia [18] or chronic liver diseases [19] in the presence of iron excess e.g. released from an increased red blood cell turnover [19,20]. An alternative explanation for the paradox response of hepatocellular hepcidin to direct iron exposure could be the fact that the actual location of "iron sensing" occurs in a different type of cells e.g., in the endothelial bed which is indirectly communicated to hepatocytes by intercellular signaling though the BMP6/SMAD pathway. Our data suggest that an EC/hepatocyte co-culture system suffice to recapitulate iron sensing as observed in vivo
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