Transient Reduction of Transferrin Saturation Contributes to Hepcidin Inhibition Independently of Erfe in Mice Treated with Erythropoietin

Introduction

Erythropoiesis is the major consumer of iron for hemoglobin synthesis. Erythroid cells release erythroferrone (ERFE), an erythropoietin (EPO) target gene, to signal their iron needs to the liver through hepcidin inhibition (Kautz et al., 2014). Since hepcidin degrades the iron exporter ferroportin, thus reducing body iron concentration, its inhibition by ERFE increases iron availability for erythroid cells. Hepcidin is mainly controlled by the BMP-SMAD-pathway. We demonstrated that ERFE inhibits hepcidin only when the BMP-SMAD signaling is attenuated (Nai et al., 2016). However, how the BMP-SMAD pathway is reduced in a physiologic context of increased erythropoiesis remains unknown. The major source of iron for erythroid cells is the circulating diferric-transferrin (holo-TF) that is uptaken by TFR1, highly expressed by erythroid cells. Since holo-TF, measured as transferrin saturation, is involved in hepcidin activation by the BMP-SMAD pathway, we hypothesized that increase iron uptake by EPO-stimulated erythropoiesis reduces transferrin saturation, thus inhibiting hepcidin earlier than and independently of ERFE.

Methods

C57BL/6 wild type and Erfe KO male mice (8-9 week-old) were challenged with a single EPO injection (200 U/mouse) and sacrificed at early time points (3-15 hrs) post-treatment. To analyze erythroid differentiation and Tfr1 levels at short time points after EPO injection (3 and 6 hrs post-treatment), Ter119+ cells were separated from bone marrow (BM) and spleen according to Forward Scatter (FSC) value and CD44 expression, as described(Liu et al., 2013). Tfr1 was measured by flow cytometry in isolated population and expressed as MFI. Expression of BM and spleen Erfe, GypA and Tfr1 was evaluated by qRT-PCR. Hamp, Id1 and Atoh8 were measured from total liver of EPO- or saline-treated mice. Serum iron, transferrin saturation (TS) and liver iron content were evaluated by standard methods (Pagani et al., 2011).

Results

Wild type mice were analyzed at different time point (3, 6, 9 and 15 hrs) after EPO injection. EPO-treated mice upregulate Erfe mRNA both in BM and spleen, starting from 3 hrs post-injection. Hepcidin and the BMP-SMAD target genes Id1 and Atoh8 are significantly decreased at 9 hrs post-EPO. Although Id1 and Atoh8 start to increase 15 hrs post-EPO, hepcidin remains low due to Erfe inhibition. EPO treatment transiently (at 6 and 9 hrs) reduces both serum iron and TS that are increased thereafter (15 hrs) due to hepcidin inhibition. Since EPO stimulates Tfr1 expression in erythroid cells, we measured Tfr1mRNA and protein levels in BM and spleen. Tfr1 mRNA is slightly increased in the spleen and shows a trend towards increase in the BM at 6 hrs post-EPO, whereas it is comparable to untreated mice at 9 and 15 hrs post-EPO. However, flow cytometry analysis shows that, despite the number of erythroid precursors expressing Tfr1 is comparable in untreated and EPO treated mice, membrane Tfr1 is strongly increased at 3 and 6 hrs post-EPO, indicating that increased erythroid uptake accounts for serum iron/TS reduction shortly after EPO treatment. To demonstrate that increased Tfr1 expression by EPO in erythroid cells downregulates the liver BMP-SMAD pathway and hepcidin through a transient reduction in circulating iron/TS and independently of Erfe, we analyzed EPO-injected Erfe KO mice. Both in wild type and Erfe KO animals Id1 is strongly inhibited at 6-9 hrs post EPO. Interestingly, Erfe KO animals show a trend towards a reduction in hepcidin at 9 hrs post-EPO, and a significant decrease at 15 hrs, with low serum iron and TS.

The mild but significant effect observed in Erfe KO mice suggests that downregulation of the BMP-SMAD pathway due to transient reduction of TS contributes to hepcidin inhibition in an erythroid-dependent, but Erfe-independent, manner.

Conclusions

EPO stimulates erythroid expression of membrane Tfr1 at short time point after treatment, causing a transient decreased in transferrin saturation and a downregulation of the hepatic BMP-SMAD pathway that contributes to hepcidin inhibition independently of Erfe.