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Erythropoiesis, circulating iron and hepcidin
brane protease, serine 6 modulate BMP/SMAD signaling and refine hepcidin production, allowing the precise regu- lation necessary to maintain iron homeostasis.4,7-9
The influence of the erythroid regulator is readily appar- ent in conditions such as b-thalassemia, in which the increased iron demands of the expanded erythroid marrow signal a decrease in hepcidin production and subsequent iron loading.10 However, the molecular mechanism by which developing erythrocytes signal their iron needs to hepatocytes remains poorly understood. One signaling mol- ecule that has recently been identified is erythroferrone, a member of the tumor necrosis factor superfamily of cytokines which is encoded by the ERFE gene.11 Erythroferrone is produced by erythroblasts11 and is detectable in the circulation following stimulated erythro- poiesis.12 Importantly, recombinant erythroferrone has been shown to inhibit hepcidin production in both primary hepa- tocytes and in mice.11 In addition, the inhibition of hepcidin by stimulated erythropoiesis is blunted in Erfe knockout mice and hepcidin expression normalizes in b-thalassemic animals lacking erythroferrone,11 providing strong evidence supporting a role for erythroferrone in hepcidin regulation.
There is clear evidence that erythroferrone can influence hepcidin expression in response to stimulated erythro- poiesis, but the level of iron in the circulation has also been proposed to be involved.13-15 Iron in the plasma is predomi- nantly bound to the protein transferrin.16 As each transferrin molecule can bind two iron atoms and physiological levels of circulating iron are not high enough to saturate all trans- ferrin binding sites, circulating transferrin can exist in four different states: apo-transferrin; two forms of monoferric transferrin; and diferric transferrin.17 Iron bound to transfer- rin is taken up by cells via receptor-mediated endocytosis following the binding of transferrin to cell surface transfer- rin receptor 1 (TFR1).16 By far the largest sink for transferrin- bound iron in the circulation is the developing erythrocytes of the bone marrow10 and, as TFR1 has the greatest affinity for the diferric isoform,16 increases in the erythropoietic rate would be expected to preferentially reduce circulating difer- ric transferrin levels. Indeed, we have previously demon- strated such a change following treatment with the hemolytic agent phenylhydrazine in rats.18 As diferric trans- ferrin has been implicated in the regulation of hepcidin expression,13,19 changes in diferric transferrin levels follow- ing a stimulus to increase erythropoiesis could augment the effect of erythroferrone and play a role in the inhibition of hepcidin production. Such a mechanism has been suggest- ed by Nai et al. to explain the reduction in hepcidin expres- sion following the administration of erythropoietin to mice.14 While Kautz et al. saw no change in serum iron lev- els in mice injected with erythropoietin, they did not specif- ically examine diferric transferrin.11 In order to determine whether diferric transferrin might contribute to the decrease in hepcidin expression that occurs following stim- ulated erythropoiesis, we examined its level in erythropoi- etin-injected mice. We also examined the effect of altering diferric transferrin levels in mice following stimulated ery- thropoiesis.
Methods
Animals
Six-week old male C57BL/6 mice obtained from the Animal Resources Centre (Perth, Australia) were used for all experiments
and were maintained on a standard rodent chow (120 mg/kg iron, Norco Stockfeed, Lismore, Australia). To stimulate erythropoiesis, mice were intravenously administered 10 U/g body weight of human erythropoietin (Epoetin alfa, Eprex, Janssen, Macquarie Park, Australia). The mice were euthanized 0, 5, 9, 12, 15 or 18 h later. Prior to euthanasia, mice were anesthetized (200 mg/kg ket- amine, 10 mg/kg xylazine) and blood was withdrawn by cardiac puncture. Blood for diferric transferrin quantitation was collected in heparin-coated tubes, briefly centrifuged, and the plasma stored at -80°C. Blood for serum iron determination was allowed to clot and the serum stored at -80°C. Following euthanasia, the liver, spleen and bone marrow were removed and snap frozen for sub- sequent analysis.
To study the effects of altering serum iron on erythropoietin- induced inhibition of hepcidin expression, four groups of mice were used. Two groups were intravenously injected with erythro- poietin (10 U/g body weight) 9 h prior to euthanasia, while the remaining two groups served as uninjected controls. Of the two groups injected with erythropoietin, one group was administered iron intravenously (2.5 mg/g body weight ferric citrate monohy- drate in 5 mM citrate buffer, pH 7.0) while the other group was injected with sodium citrate (equimolar with the citrate in the fer- ric citrate solution) in 5 mM citrate buffer as a control. Similar injections were administered to the two groups not given erythro- poietin. All ferric citrate or sodium citrate injections were given 4 h prior to euthanasia. To confirm that the injected iron led to an increase in circulating diferric transferrin, additional groups of mice were euthanized 5 min after the iron or citrate injections and blood was collected for analysis.
To control for circadian variations in serum iron levels and the expression of Hamp1, the gene encoding hepcidin, all experimen- tal animals were euthanized between 10:00 am and 12:30 pm local time. We did not observe any consistent alterations in hepatic Hamp1 expression during this period.
Male mice were used in all studies as differences in the absolute levels of some iron parameters have been reported in males and females.20 However, the mechanisms regulating iron homeostasis and hepcidin expression are thought to be similar in both genders. All animal experiments were approved by the QIMR Berghofer Animal Ethics Committee.
Analysis of blood and tissue samples
Details of the analysis of serum iron levels, liver iron concentra- tion and gene and protein expression are included in the Online Supplementary Methods.
Statistics
All results are expressed as mean ± standard error of the mean (SEM). Statistical differences between groups were calculated using ANOVA followed by either Tukey post hoc testing for sam- ples with equal variance or Games-Howell post hoc testing for sam- ples with unequal variance. IBM SPSS Statistics version 22 soft- ware (IBM Australia, St Leonards, Australia) was used. A P value of less than 0.05 was considered statistically significant.
Results
Reduced hepatic Hamp1 expression is associated with increased splenic and bone marrow Erfe production following injection of erythropoietin
Previous studies in mice have implicated erythroferrone in the inhibition of hepcidin production following admin- istration of erythropoietin.11,12 Other factors, such as the level of diferric transferrin in the circulation, might also
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