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C.S.G. Mirciov et al.
AB
CD
Figure 8. Tfr1 expression 4 hours after iron injection in erythropoietin-treated mice. Six-week old male C57BL/6 mice were injected intravenously with 10 U/g body weight human erythropoietin. Five hours later, mice were intra- venously injected with either 2.5 mg/g body weight ferric citrate or an equimo- lar amount of citrate as sodium citrate. Mice were euthanized 4 h after this final injection and tissues were taken for analysis. Splenic Tfr1 expression (A, B) and bone marrow Tfr1 expression (C, D) were determined for each group. Gene expression levels were calculated relative to either the general house- keeping gene Hprt or the erythroid-spe- cific marker Gypa and are expressed as a proportion of the values in mice injected with sodium citrate but not with erythropoietin. The data represent the mean ± SEM with the number of mice in each group indicated in paren- theses along the x-axis. Con: control mice injected with sodium citrate; Fe: mice injected with ferric citrate; No Epo: mice that were not injected with erythropoietin; Epo: mice that were injected with erythropoietin. *P<0.05; ***P<0.005.
spleen. In fact, Erfe expression in both tissues was higher in mice that received both erythropoietin and iron com- pared to those that received erythropoietin only, although the changes were not significant. However, as spleen size was also increased, it is highly likely that circulating ery- throferrone levels are higher in erythropoietin-treated ani- mals injected with iron, implying that increased serum iron levels can overcome the effect of erythroferrrone, at least in the early stages of erythroid stimulation. However, this does not always occur. For example, we observed a significant increase in diferric transferrin levels 18 h after erythropoietin injection despite Hamp1 expression being at its lowest level at this time. While the reason for this is unclear, it is possible that circulating erythroferrone levels increased over the time course. While no such change was observed in Erfe message levels, a previous study showed that Erfe mRNA expression does not always correspond to serum erythroferrone levels.12 Another possible explana- tion is that the increase in diferric transferrin seen later in our time course had not had sufficient time to influence Hamp1 expression, as changes in serum iron levels can take several hours to exert an effect on hepcidin produc- tion.15
tion.31 This latter finding is consistent with that of an earlier study (carried out before hepcidin was identified) showing that injected transferrin was able to suppress iron absorp- tion in hypotransferrinemic mice in which erythropoiesis had been normalized.32 However, as transferrin levels sub- sequently declined, iron absorption increased. Secondly, although NTBI in the form of ferric citrate was adminis- tered to the mice in the current study, it had previously been demonstrated that any NTBI formed in this way is removed very rapidly from the circulation with a half-life of <30 s.23 In contrast, we have shown that diferric trans- ferrin levels remain elevated 4 h after iron injection (Figure 5D). Thirdly, as mentioned previously, a mechanism for the detection of diferric transferrin levels in the circulation and the subsequent regulation of Hamp1 expression has been proposed and is supported by in vivo evidence,33,34 whereas no such mechanism has been described for NTBI. Finally, these results are consistent with multiple cell cul- ture studies showing that treatment of cells with iron salts does not directly stimulate hepcidin production.35,36 In fact, Hamp expression could only be stimulated when transfer- rin-bound iron was supplied.36 Therefore, despite not being able to definitively exclude a role for NTBI, it is far more likely that diferric transferrin is the major stimulator of Hamp1 expression in our studies.
The increase in Hamp1 expression occurred despite sig- nificant increases in Erfe expression in the bone marrow or
Certain mouse models of b-thalassemia exhibit high transferrin saturation yet continue to load with iron due to low hepcidin levels.37 It is likely that, in these instances, the expansion of the erythroid marrow due to chronic
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haematologica | 2018; 103(10)