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C.S.G. Mirciov et al.
play a role. To examine this, we established a model of erythropoietin-induced hepcidin inhibition. In this model, we ensured that all mice were euthanized within a narrow window of time to ensure that known circadian variations in both hepatic Hamp1 expression and serum iron levels21,22 did not influence the results.
In agreement with findings of previous studies,11,14 hepatic Hamp1 levels progressively decreased following erythropoietin administration, with significant inhibition first observed after 9 h (Figure 1A). By 18 h, Hamp1 expression was only 12% of the control value. Only minor changes in spleen weight were observed (Figure 1B), pos- sibly because the time points examined were too soon after erythropoietin injection for significant increases in the number of erythroid precursors to have occurred. Despite this, a large increase in Erfe expression (>86-fold) was seen in both the spleen (Figure 1C, D) and bone mar- row (Figure 1E, F) of injected animals at the 5 h time point. This increase in Erfe expression was observed at the 5, 9, 12 and 15 h time points, but splenic levels had reduced by 18 h after erythropoietin injection. Normalizing Erfe expression to the general housekeeper hypoxanthine gua- nine phosphoribosyl transferase (Hprt) or the erythroid precur- sor specific marker glycophorin A (Gypa) did not alter the expression pattern seen. Our results agree with those of
previous studies11,14 and confirm that our model can be used to examine the potential role of diferric transferrin in erythropoietin-induced hepcidin inhibition.
Erythropoietin administration increases Tfr1 expres- sion in the bone marrow and spleen and causes a transient decrease in circulating diferric transferrin levels
Developing erythrocytes are the major sink for circulat- ing iron and this iron demand is increased when erythro- poiesis is stimulated.10 To determine how rapidly this increase in demand occurs, we examined the expression of the Tfr1 gene, the product of which is central to the main cellular iron import pathway in developing erythro- cytes.10 In the bone marrow, Tfr1 expression increased rap- idly after erythropoietin administration, showing maxi- mal expression at 5 h when normalized to either Hprt (Figure 2A), or the erythroid-specific housekeeper, Gypa (Figure 2B). The increase in splenic Tfr1 was not as pro- nounced, with significant increases only observed when its levels were normalized to those of Gypa (Figure 2C,D). These results indicate that there is a very rapid increase in the iron demands of developing erythroid cells following erythropoietin administration, and imply that iron is removed from the circulation more rapidly than normal,
ABC
DEF
Figure 1. Hamp1 and Erfe expression following erythropoietin injection in mice Six-week old male C57BL/6 mice were euthanized 0, 5, 9, 12, 15 or 18 h following the intravenous injection of 10 U/g body weight human erythropoietin and tissues were taken for analysis. Hepatic Hamp1 expression (A), spleen weight (B), splenic Erfe expression (C, D) and bone marrow Erfe expression (E, F) were determined for each time point. Gene expression levels were calculated relative to either the gen- eral housekeeping gene Hprt or the erythroid specific marker Gypa, and are expressed as a proportion of the values at 0 h. The data represent the mean ± SEM with the number of mice in each group indicated in parentheses along the x-axis. Statistical significance is shown relative to the 0 h group. *P<0.05; **P<0.01; ***P<0.005.
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haematologica | 2018; 103(10)