Contributions
Abstract: S275
Type: Oral Presentation
Session title: Focus on iron metabolism
Background
Iron availability is essential for erythropoiesis and needs to be maintained within the physiological range to prevent hematological disorders. The liver hormone hepcidin is the master regulator of systemic iron homeostasis. It binds and triggers degradation of the sole cellular iron exporter ferroportin (Fpn), blocking dietary iron absorption and iron release from cellular stores. Hepatic iron levels control hepcidin mRNA expression in hepatocytes (HCs) by modulating the BMP signaling pathway. Conditions of iron overload induce the growth factor BMP6 that is released from liver sinusoidal endothelial cells (LSECs), the most relevant cell type of the liver for sensing of iron availability.LSECs account for only a very small percentage of total liver cells and primary cultures are difficult to generate as these cells easily transdifferentiate. Therefore detailed molecular insights into how LSECs sense iron levels and the signaling pathways controlling BMP6 production in response to iron levels are challenging to obtain.
Aims
Our aim is to understand the molecular mechanisms of how the liver senses iron availability, controls BMP6 production and hepcidin synthesis. Results are expected to be translated to new therapeutic strategies for iron-related disorders.
Methods
Murine liver cell suspensions were prepared by in vivo liver perfusion and digestion. HCs and LSECs were isolated combining cell gradient isolation and exclusion adherence steps or immunoselection with antibodies conjugated to magnetic beads. The quality of the LSEC preparations was assessed by FACS analysis and Scanning Electron Microscopy. Ferritin protein levels and Transferrin Receptor 1 mRNA expression served as markers to determine the intracellular iron content of isolated liver cells. RNA-seq of HCs and LSECs isolated from FpnC326S mice was performed with Illumina NextSeq500.
Results
We established highly pure primary murine LSEC cultures with 95% of cells positive for LSEC markers. Analyses of the LSEC ultrastructure indicated the presence of fenestrae - a unique LSEC feature that confirms the absence of transdifferentiation. We show that primary LSECs fail to modulate BMP6 expression in response to iron treatment, but require a co-culture with iron-loaded primary HCs to significantly increase BMP6 expression. These data are supported by findings from a genetic mouse model of systemic iron overload (FpnC326S), hallmarked by iron-loaded HCs and elevated BMP6 levels despite a lack of increased iron content in LSECs. RNA-seq analysis of primary HCs and LSECs isolated from FpnC326S mice identified altered signaling pathways. Experiments are ongoing to combine these results with in silico ligand-receptor interaction analysis and experiments in primary cell cultures to unravel the hepatocytic factor essential for the iron-dependent regulation of BMP6.
Conclusion
We established a high quality preparation of primary LSECs as a fundamental tool to investigate how these cells sense iron to modulate BMP6 expression. Our ex vivo data reveal that, contrary to former belief, the LSEC-secreted growth factor BMP6 is not regulated by changes in the LSEC intracellular iron content. Accordingly, we show that LSECs isolated from a mouse model of iron overload are not iron-loaded, despite increased BMP6 expression. Importantly, co-culture of primary LSECs with HCs demonstrates that the latter are essential to induce BMP6 expression when iron levels are increased. These data support a model in which cell-to-cell communication between HCs and LSECs is essential to sense iron availability and activate BMP6 transcription.
Keyword(s): Endothelial cell, Hepatocyte, Hepcidin, Iron overload
Abstract: S275
Type: Oral Presentation
Session title: Focus on iron metabolism
Background
Iron availability is essential for erythropoiesis and needs to be maintained within the physiological range to prevent hematological disorders. The liver hormone hepcidin is the master regulator of systemic iron homeostasis. It binds and triggers degradation of the sole cellular iron exporter ferroportin (Fpn), blocking dietary iron absorption and iron release from cellular stores. Hepatic iron levels control hepcidin mRNA expression in hepatocytes (HCs) by modulating the BMP signaling pathway. Conditions of iron overload induce the growth factor BMP6 that is released from liver sinusoidal endothelial cells (LSECs), the most relevant cell type of the liver for sensing of iron availability.LSECs account for only a very small percentage of total liver cells and primary cultures are difficult to generate as these cells easily transdifferentiate. Therefore detailed molecular insights into how LSECs sense iron levels and the signaling pathways controlling BMP6 production in response to iron levels are challenging to obtain.
Aims
Our aim is to understand the molecular mechanisms of how the liver senses iron availability, controls BMP6 production and hepcidin synthesis. Results are expected to be translated to new therapeutic strategies for iron-related disorders.
Methods
Murine liver cell suspensions were prepared by in vivo liver perfusion and digestion. HCs and LSECs were isolated combining cell gradient isolation and exclusion adherence steps or immunoselection with antibodies conjugated to magnetic beads. The quality of the LSEC preparations was assessed by FACS analysis and Scanning Electron Microscopy. Ferritin protein levels and Transferrin Receptor 1 mRNA expression served as markers to determine the intracellular iron content of isolated liver cells. RNA-seq of HCs and LSECs isolated from FpnC326S mice was performed with Illumina NextSeq500.
Results
We established highly pure primary murine LSEC cultures with 95% of cells positive for LSEC markers. Analyses of the LSEC ultrastructure indicated the presence of fenestrae - a unique LSEC feature that confirms the absence of transdifferentiation. We show that primary LSECs fail to modulate BMP6 expression in response to iron treatment, but require a co-culture with iron-loaded primary HCs to significantly increase BMP6 expression. These data are supported by findings from a genetic mouse model of systemic iron overload (FpnC326S), hallmarked by iron-loaded HCs and elevated BMP6 levels despite a lack of increased iron content in LSECs. RNA-seq analysis of primary HCs and LSECs isolated from FpnC326S mice identified altered signaling pathways. Experiments are ongoing to combine these results with in silico ligand-receptor interaction analysis and experiments in primary cell cultures to unravel the hepatocytic factor essential for the iron-dependent regulation of BMP6.
Conclusion
We established a high quality preparation of primary LSECs as a fundamental tool to investigate how these cells sense iron to modulate BMP6 expression. Our ex vivo data reveal that, contrary to former belief, the LSEC-secreted growth factor BMP6 is not regulated by changes in the LSEC intracellular iron content. Accordingly, we show that LSECs isolated from a mouse model of iron overload are not iron-loaded, despite increased BMP6 expression. Importantly, co-culture of primary LSECs with HCs demonstrates that the latter are essential to induce BMP6 expression when iron levels are increased. These data support a model in which cell-to-cell communication between HCs and LSECs is essential to sense iron availability and activate BMP6 transcription.
Keyword(s): Endothelial cell, Hepatocyte, Hepcidin, Iron overload