The role of the liver serine protease TMPRSS6 in iron homeostasis

Hepcidin the main controller of iron homeostasis reduces plasma iron levels by degrading iron exporter ferroportin in enterocytes and macrophages. Hepcidin regulation is the result of a balance between its inhibition and activation. The inhibition is due to the liver serine protease TMPRSS6. The activation is promoted by Bone-Morphogenetic-Protein 6 (BMP6) that interacting with BMP receptors and their coreceptor Hemojuvelin (HJV) activates BMP-SMAD pathway. Hepatic TFR2 (Transferrin Receptor 2) and HFE upregulate hepcidin (HAMP) transcription through a still undefined pathway. In vitro TMPRSS6 cleaves membrane-HJV, a process that releases soluble fragments in the culture media. In the first paper, to better characterize the serine protease activity, we investigated the HJV cleavage sites by mutagenizing the potential targets arginine (R) to alanine. We analyzed the processing of HJV R mutants and the pattern of fragments released in culture media by TMPRSS6. Abnormal fragments were observed for mutants at R121, R176, R218, R288 and R326 and residues 121 and 326 were identified as TMPRSS6 cleavage sites. HJV belongs to Repulsive-Guidance-Molecules (RGM) family, undergoes an autoproteolytic process and is exported on cell surface both as heterodimeric and full-length isoforms. Using a N-terminal-FLAG-tagged HJV we showed that R121 is critical in the rearrangement of the N-terminal heterodimeric HJV. Exploiting the recently reported RGMb crystallographic structure we generated a HJV model that was used as an input structure for all-atoms molecular dynamics simulation in explicit solvent. By in silico studies we concluded that some mutants appear TMPRSS6 insensitive, likely because of structural destabilization. In the second paper we focused on the regulation of Bmp6 expression. Bmp6 is modulated by iron but the molecular mechanisms and the cell type involved remain uncertain. Since Bmp6 is produced by both hepatocytes (HCs) and non-parenchymal cells (NPCs), we optimized a method to isolate liver cells population (HC, sinusoidal endothelial cells (LSEC) and Kupffer cells) to define cells involved in Bmp6 regulation. We investigated Bmp6 transcription both in wild type mice whose diet was modified in iron content in acute (1 day) or chronic (3 weeks) manner and in disease models of iron deficiency (Tmprss6-/- mouse) and iron overload (Hjv-/- mouse). After 1-day diet that induces a mild iron increase, only HCs and Kupffer cells modulate Bmp6 while LSECs are unresponsive. When hepcidin expression is abnormal in iron overload (Hjv-/-) and deficiency (Tmprss6-/-), Bmp6 expression in NPCs is not related to intracellular iron but parallels iron uptake. In particular Bmp6 transcription is low in Tmprss6-/-, although Tfr1 expression suggests increased iron in NPCs, due to high hepcidin levels. This defines a paracrine mechanism of hepcidin increase through Bmp6 by NPCs. Modulating hepcidin they control systemic iron homeostasis and promote their own iron retention/release as a protective mechanism towards HC iron excess/deficiency. This mechanism is lost in disease models of hepcidin deregulation. TMPRSS6 inactivation causes Iron-Refractory-Iron-Deficiency-Anemia due to inappropriately high hepcidin levels, while mutation in TFR2 causes hereditary hemochromatosis. To evaluate whether Tmprss6 is implicated in the modulation of the Tfr2-dependent Hamp expression, in the third paper we generated the total Tfr2-/-Tmprss6-/- mice and the liver-specific Tfr2 knock-out (Tfr2LCKO) Tmprss6-/- mice. Both models have increased hepcidin levels and iron-deficiency anemia like Tmprss6-/-, proving that Tmprss6 acts downstream Tfr2. These results identify Tmprss6 cleavage sites of hemojuvelin, verify the mechanism of iron handling in liver NPCs in Tmprss6-/- and establish that Tmprss6 is downstream Tfr2, possibly regulated by Tfr2 itself.