Session: 102. Regulation of Iron Metabolism
Hematology Disease Topics & Pathways:
Diseases, Genetic Disorders, Biological Processes, iron metabolism, pathways
Hepcidin, the liver hormone that negatively controls body iron levels, is a BMP-SMAD target gene. Its activation requires the formation of a multiprotein complex composed of BMP ligands (BMP2 and BMP6), type I (ALK2 and ALK3) and type II BMP receptors, as well as regulatory proteins such as HJV, TFR2 and HFE. Mutations in those genes cause Hereditary Hemochromatosis, a disorder characterized by low hepcidin levels and iron overload. Hepcidin is inhibited by the transmembrane serine protease TMPRSS6 and the immunophilin FKBP12 (Colucci, 2017). Although the mechanism of action of TMPRSS6 has been identified (Silvestri, 2008), how exactly FKBP12 inhibits hepcidin is still not fully understood.
The aims of this study are:
- To investigate the mechanism of BMP-SMAD pathway regulation by FKBP12, focusing on BMP type I homo- and hetero-dimerization, their interaction with type II receptors and the response to ligands;
- To provide the proof of principle that FKBP12 downregulation/sequestration by antisense oligonucleotides (ASOs) or by pharmacologic approaches, respectively, can be a potential therapeutics for low hepcidin-iron overload disorders.
Materials and methods
The role of FKBP12 and its sequestration by Tacrolimus (TAC) in regulating the interaction between type-I and -II BMP receptors were explored by co-immunoprecipitation. Ligand responsiveness was assessed by SMAD1/5/8 phosphorylation and BMP Responsive Element (BRE)-luciferase assay. Murine primary hepatocytes (mHCs) were subjected to RNAi of Alk2 or Alk3 and treated with TAC to assess their role in TAC-dependent hepcidin activation. Hjv-KO mice were treated with TAC (0.37µg/hr for 28 days) via mini osmotic pumps. Fkbp12 downregulation in wild-type (WT) and Hjv-KO mice was induced by treatment with specific ASOs (50 mg/kg twice a week for 6 weeks). Fkbp12 and Tmprss6 were downregulated in hepatocytes by GalNac-conjugated ASOs (5 and 1.25 mg/kg respectively). Mice were sacrificed and analyzed for iron parameters, CBC, erythropoiesis, hepcidin and BMP-SMAD target gene (Id1) expression.
The immunosuppressive drug TAC upregulates hepcidin both in hepatoma cells and with acute treatment in vivo (Colucci, 2017). To better understand the mechanism, mHCs were silenced for Alk2, Alk3 or both and treated with TAC. Alk2, but not Alk3 silencing completely abrogated hepcidin upregulation by TAC. Since TAC sequesters FKBP12, we conclude that FKBP12 binds and regulates ALK2, but not ALK3 in mHCs. FKBP12 sequestration by TAC stabilized ALK2 homodimers and favored the interaction with type II BMP receptors, thus driving the formation of active tetrameric receptor complexes. Accordingly, TAC treatment synergized with BMP6-dependent BMP-SMAD pathway activation, not only in vitro but also in vivo as assessed by hepcidin upregulation in Hjv-KO mice chronically treated with TAC. Liver Fkbp12 downregulation by ASOs activated the BMP-SMAD pathway both in WT and Hjv-KO mice, as indicated by Id1 upregulation, but hepcidin expression remained unchanged. Fkbp12 was also reduced in the spleen of Fkbp12 ASO-treated mice, unexpectedly increasing the percentage of early erythroid precursors and the expression of erythroferrone (Erfe), the hepcidin inhibitor. To avoid the effect on erythropoiesis, we evaluated GalNAc-conjugated Fkbp12 and control ASOs in WT mice. Surprisingly, hepatocyte Fkbp12 downregulation by GalNac-Fkbp12 ASOs increased Tmprss6 expression, likely as a compensatory feedback loop to decrease hepcidin activation. Accordingly, a partial Tmprss6 downregulation with GalNAc-Tmprss6 ASO in combination with GalNAc-Fkbp12 ASO synergistically upregulated hepcidin expression.
- In mHCs FKBP12 binds and regulates ALK2 and not ALK3;
- FKBP12 sequestration by TAC activates the BMP-SMAD pathway by favoring the formation of active BMP type I and type II tetrameric complexes and increasing the sensitivity to the BMP6 ligand;
- Fkbp12 plays a role in erythropoiesis. Its downregulation in the spleen of ASO-treated mice increases immature erythroid precursors and the production of the hepcidin inhibitor Erfe, that likely counteracts the ASO-mediated effect on hepcidin upregulation in the liver;
- Fkbp12 downregulation in hepatocytes increases Tmprss6 expression in mice, whereas reducing expression of both inhibitors with combined ASO treatment synergistically upregulates hepcidin expression.
Disclosures: Aghajan: Ionis Pharmaceuticals, Inc.: Current Employment. Guo: Ionis Pharmaceuticals, Inc.: Current Employment.
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