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Erythropoiesis, circulating iron and hepcidin
be able to regulate HAMP expression in an individual with HFE-related hemochromatosis, although it could still be regulated through other pathways. An additional detec- tion mechanism involving TFR2, a TFR1 homolog, has also been described.13 Although the precise pathways by which these molecules signal changes in hepcidin produc- tion remains unclear, their involvement in detecting difer- ric transferrin levels is widely accepted.3,10,27,28 While show- ing a correlation only, when viewed in conjunction with the evidence supporting diferric transferrin as a regulator of hepcidin, the current study suggests that diferric trans- ferrin levels are likely to contribute to the initial reduction in Hamp1 expression seen following an erythropoietic stimulus. However, whether the decrease in diferric trans- ferrin levels is required for the inhibition of hepcidin is unclear. Erfe expression was also increased at the 5 h time point, although the lack of a reliable commercial assay for mouse erythroferrone made it difficult to determine the amount of functional erythroferrone at this time. Further research is required to determine whether both stimuli are required or whether there is a degree of redundancy in the pathways inhibiting hepcidin at these early time points. In contrast, serum iron is clearly not involved in reducing Hamp1 expression towards the end of the time course, as
serum iron parameters quickly returned to normal levels and even exceeded control values at these later time points.
Our results also show that increasing serum iron levels soon after erythropoietin injection can overcome the inhibitory effect of stimulated erythropoiesis and prevent any decrease in Hamp1 expression. While our data suggest that changes in diferric transferrin levels are the likely cause, a role for non-transferrin-bound iron (NTBI) cannot be excluded, as this is the form of iron injected in our stud- ies. However, there are several reasons why a major role for NTBI in hepcidin regulation is unlikely. Firstly, studies using the hypotransferrinemic mouse suggest that it is the transferrin-bound iron in the circulation that is important for hepcidin regulation. These mice have greatly reduced transferrin levels and, as a consequence, high levels of NTBI.29,30 With limited transferrin, iron supply to the mar- row is compromised and the resulting anemia reduces hepcidin production. So hepcidin levels are low, despite very high levels of NTBI.29 Even in hypotransferrinemic mice subjected to myeloablation to remove the effect of stimulated erythropoiesis, hepcidin expression could only be increased if transferrin was administered, making it unlikely that NTBI has a major effect on hepcidin produc-
ABC
DE
Figure 7. Erfe expression and spleen weight 4 hours after iron injection in erythropoietin-treated mice. Six-week old male C57BL/6 mice were injected intravenous- ly with 10 U/g body weight human erythropoietin. Five hours later, mice were intravenously injected with either 2.5 mg/g body weight ferric citrate or an equimolar amount of citrate as sodium citrate. Mice were euthanized 4 h after this final injection and various tissues were taken for analysis. Splenic Erfe expression (A, B), bone marrow Erfe expression (C, D) and spleen weight (E) were determined for each group. Gene expression levels were calculated relative to either the general housekeeping gene Hprt or the erythroid-specific marker Gypa and are expressed as a proportion of the values in mice injected with sodium citrate but not with ery- thropoietin. The data represent the mean ± SEM with the number of mice in each group indicated in parentheses 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.
haematologica | 2018; 103(10)
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