Hepatocyte Iron Content Controls BMP6-Dependent Hepcidin Regulation

Liver is a heterogeneous organ central for the control of systemic iron homeostasis. Among the different cell types, only hepatocytes (parenchymal cells) produce the iron regulatory hormone hepcidin. Hepcidin binds to the iron exporter ferroportin and triggers its degradation , thus modulating iron absorption from the diet and iron release from macrophagesdistrib. The iron-mediated hepcidin response is controlled via the SMAD1/5/8 pathway which is activated by bone morphogenetic proteins (BMPs). In contrast to hepcidin, BMPs are mainly produced by liver non-parenchymal cells, questioning the exclusive role of hepatocytes in regulating iron homeostasis. In particular, liver sinusoidal endothelial cells (LSECs) are emerging as cells that sense iron availability and modulate hepcidin levels through the production of BMP2 and BMP6 - the main regulators of hepcidin. However, how LSECs sense iron and which signals control BMP2 and BMP6 production have to be clarified.

We have generated a mouse model with heterozygous depletion of the ferroportin allele (Slc40a1^wt/trp). These mice show normal haematological parameters, serum iron levels and transferrin saturation. However, the liver is iron deficient and expresses low levels of hepcidin. The phosphorylation of SMAD1/5/8 proteins is lower in Slc40a1^wt/trp compared to wild type littermates, explaining the hepcidin phenotype. To further investigate the molecular mechanism underlying hepcidin downregulation mediated by hepatic iron deficiency, we established a protocol to isolate hepatocytes and LSECs from total mouse liver and analyzed expression of iron-related genes. BMP6 is downregulated in LSECs of Slc40a1^wt/trp mice, explaining the diminished activity of the SMAD1/5/8 signaling pathway. We next examined intracellular iron levels in hepatocytes and LSECs by measuring ferritin (Ft) and transferrin receptor 1 expression (Tfr1), two genes whose expression is regulated via the IRE/IRP system according to intracellular iron stores. Hepatocytes have an iron-deficient molecular signature, with low levels of Ft and a high expression of Tfr1. Surprisingly in LSECs both Ft and Tfr1 are unchanged, suggesting that this cellular population has unaltered iron levels.

Taken together, our results show for the first time that decreased hepatic iron content is self-sufficient to cause a dramatic reduction in hepcidin expression and secretion. The hepcidin downregulation in the Slc40a1^wt/trp mouse model is caused by a reduction in BMP6 levels that correlates with hepatocyte iron content. BMP6 modulation cannot be explained either by differences in circulating iron or by altered intracellular iron levels of LSECs, suggesting that BMP6 is not regulated by LSEC-mediated iron sensing. In our mouse model BMP6 seems to be linked with the intracellular iron content of hepatocytes. Therefore, we hypothesize that hepatocytes are the sensor of liver iron content which controls BMP6 expression in LSECs. RNAseq analysis on hepatocytes from Slc40a1^wt/trp mice has been performed to identify underlying mechanisms.