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C.S.G. Mirciov et al.
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iron injections had no effect on total iron binding capacity, a measure of total transferrin levels (Figure 4C). The analy- sis of transferrin species showed that transferrin was almost fully saturated 5 min after iron injection, with no apo-transferrin detectable (Figure 4D). In agreement with our earlier observations (Figure 3D), a decrease in diferric transferrin was detected in mice injected with erythropoi- etin and sodium citrate when compared to those injected with sodium citrate alone.
A second cohort of mice was euthanized 4 h after iron injection (9 h after erythropoietin injection). Total serum iron and transferrin saturation in these iron-injected mice were lower than those observed at the 5 min time point (Figure 4A,B), with significant differences seen only in the groups injected with erythropoietin (Figure 5A,B). Once again, the injections had no influence on total iron binding capacity (i.e. total transferrin levels) (Figure 5C). Diferric transferrin levels remained significantly elevated 4 h after iron injection (Figure 5D), although the levels were reduced when compared with those 5 min after injection (Figure 4D). An analysis of liver tissue showed no change in hepatic iron levels in the groups treated with iron (Figure 5E), indicating that, although iron had been removed from the circulation in the 4 h since the iron injection, the amounts were not enough to influence stor- age iron levels.
Increasing serum iron levels can overcome the inhibitory effect of erythropoietin injection on Hamp1 expression without altering Erfe production
The effect of increasing serum iron levels on Hamp1 expression was examined in our erythropoietin-treated
Figure 3. Circulating iron parameters 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 blood was taken for analysis. Total serum iron (A), transferrin saturation (B), total iron binding capacity (C) and relative diferric transferrin levels (D) were deter- mined for each time point. The relative diferric transferrin levels represent the percentage of transferrin in the diferric form expressed as a pro- portion 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. Apo: apotransferrin; Mono: monoferric transferrin; Di: diferric transferrin. Statistical significance is shown relative to the 0 h group. *P<0.05.
mice sacrificed 4 h after iron injection. We found that increasing serum iron was able to overcome the inhibitory effect of stimulated erythropoiesis, with Hamp1 expres- sion in mice injected with both erythropoietin and iron remaining at levels similar to those in mice not injected with erythropoietin (Figure 6A). The level of phosphory- lated SMAD1/5/8 was decreased in the erythropoietin- injected mice and increased with subsequent iron injection (Figure 6B); this finding supports those of previous studies indicating that the SMAD pathway is involved in the reg- ulation of Hamp1 expression by both circulating erythro- ferrone and iron levels.14,15,24
Despite Hamp1 expression returning to normal follow- ing iron injection, splenic and bone marrow Erfe levels remained elevated in response to erythropoietin (Figure 7A-D). We also observed a significant increase in spleen weight in the erythropoietin-injected group receiving iron, possibly indicating an increase in erythroblast prolifera- tion and implying that erythropoiesis is iron restricted in the hours following an erythropoietic stimulus (Figure 7E). Consistent with this, we detected an increase in the expression of Tfr1 (normalized to Hprt) (Figure 8A) in the spleen of mice treated with both erythropoietin and iron when compared to those treated with erythropoietin alone, although the increase failed to reach statistical sig- nificance (P=0.089). A similar non-significant increase in splenic Erfe/Hprt was also seen (Figure 7A). In both cases, the difference was lost when gene expression was normal- ized to Gypa (Figures 7B and 8B), indicating that the changes were due to an increase in erythroblast number rather than individual cell expression. In contrast, we detected a decrease in Tfr1 expression in the bone marrow
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haematologica | 2018; 103(10)