Is Tmprss6 Required for Hepcidin Inhibition By Erythroferrone?

Introduction

Hepcidin, the main regulator of iron homeostasis, is inhibited when erythropoiesis is expanded. Several candidates, as GDF15 and TWSG1, have been proposed to mediate this effect but their role remains unproven. Recently, erythroferrone (ERFE), a member of the C1q/tumors necrosis factor-related protein family, has been identified as a new hepcidin inhibitor (Kautz et al., 2014). ERFE is an erythropoietin (EPO) target gene produced by bone marrow and spleen erythroblasts in conditions of stress erythropoiesis, as after bleeding or EPO treatment, and in ineffective erythropoiesis, as in beta thalassemia. Inhibiting hepatocyte hepcidin, ERFE coordinates erythroid differentiation with iron availability. In beta-thalassemia Hbb^th3/+ mice, inactivation of Erfe partially reduces liver iron content, suggesting that increased Erfe production contributes to thalassemia iron overload (Kautz et al., 2014). Here we analyzed spleen Erfe expression in models of low (iron loaded Hjv^-/- and Tfr2^-/- mice) and high (iron deficient, Tmprss6^-/- mice) hepcidin, in secondary iron overload (Hbb^th3/+ mice), in Hbb^th3/+ Tmprss6^-/- and Tfr2^-/- Tmprss6^-/- double mutants and in mice with a diet-induced iron deficiency.

Methods

Mice were maintained in accordance with the European Union guidelines. The study was approved by the IACUC of San Raffaele Scientific Institute, Milan, Italy. Hjv^-/-, Tfr2^-/-, Tmprss6^-/- and double mutant (Tfr2^-/- Tmprss6^-/- or Hbb^th3/+ Tmprss6^-/-) adult male mice were studied. A group of adult wild type mice was maintained an iron-deficient diet (ID, <3 mg/kg iron) for 3 weeks. Appropriate controls were studied. Gene expression levels were measured by quantitative real-time-PCR. Hematological and iron parameters and serum erythropoietin were studied using standard procedures.

Results

We confirm that Erfe is increased in the spleen of Hbb^th3/+ mice, characterized by anemia, ineffective erythropoiesis, high EPO, low hepcidin and iron overload. Erfe is upregulated also in Tmprss6^-/- iron deficient animals, consistent with their increased serum Epo. However, their high hepcidin levels suggest that Tmprss6 is indispensable for Erfe-mediated hepcidin inhibition. Consistent with this interpretation, in Hbb^th3/+Tmprss6^-/- double mutant mice, in which ineffective erythropoiesis and anemia are partially rescued (Nai et al., 2012), hepcidin levels are higher than in Hbb^th3/+ and comparable to those of Tmprss6^-/- mice, although Erfe remains high and serum Epo levels are similarly increased in all the three genotypes (Tmprss6^-/-, Hbb^th3/+, Hbb^th3/+Tmprss6^-/-). To further confirm the need of Tmprss6 for Erfe function, in diet-induced iron deficient animals, in which Tmprss6 is supposed to be active, Erfe expression is increased and hepcidin strongly downregulated.

In the spleen of Hjv^-/- and Tfr2^-/- mice, the expression of the erythroid markers Tfr1 and Glycophorin A (Gypa) is decreased, suggesting that splenic erythropoiesis is reduced in iron overload. In agreement Erfe is downregulated in Tfr2^-/- and mildly decreased in Hjv^-/- mice. Genetic inactivation of Tmprss6 in Tfr2^-/- mice enhances Erfe, Tfr1 and Gypa expression and serum Epo to levels comparable to Tmprss6^-/- mice and increases hepcidin although at levels lower than those found in Tmprss6^-/-.

Conclusions

Erfe upregulation in iron deficiency indicates that it is a general mediator of hepcidin inhibition. In Tmprss6^-/- mice, notwithstanding Erfe upregulation, hepcidin levels are not suppressed, suggesting that Erfe acts upstream Tmprss6, although results in the double mutant Tfr2^-/-Tmprss6^-/- require further studies. In disease models of iron overload Erfe expression is downregulated, consistent with decreased splenic erythropoiesis. The mechanisms of hepcidin inhibition by Erfe still remain to be investigated.