Abstract
Expression of hepcidin, the hormone regulating iron homeostasis, is increased by iron overload and decreased by accelerated erythropoiesis or iron deficiency. The purpose of the study was to examine the effect of these stimuli, either alone or in combination, on the main signaling pathway controlling hepcidin biosynthesis in the liver, and on the expression of splenic modulators of hepcidin biosynthesis. Liver phosphorylated SMAD 1 and 5 proteins were determined by immunoblotting in male mice treated with iron dextran, kept on an iron deficient diet, or administered recombinant erythropoietin for four consecutive days. Administration of iron increased liver phosphorylated SMAD protein content and hepcidin mRNA content; subsequent administration of erythropoietin significantly decreased both the iron-induced phosphorylated SMAD proteins and hepcidin mRNA. These results are in agreement with the recent observation that erythroferrone binds and inactivates the BMP6 protein. Administration of erythropoietin substantially increased the amount of erythroferrone and transferrin receptor 2 proteins in the spleen; pretreatment with iron did not influence the erythropoietin-induced content of these proteins. Erythropoietin-treated iron-deficient mice displayed smaller spleen size in comparison with erythropoietin-treated mice kept on a control diet. While the erythropoietin-induced increase in splenic erythroferrone protein content was not significantly affected by iron deficiency, the content of transferrin receptor 2 protein was lower in the spleens of erythropoietin-treated mice kept on iron-deficient diet, suggesting posttranscriptional regulation of transferrin receptor 2. Interestingly, iron deficiency and erythropoietin administration had additive effect on hepcidin gene downregulation in the liver. In mice subjected both to iron deficiency and erythropoietin administration, the decrease of hepcidin expression was much more pronounced than the decrease in phosphorylated SMAD protein content or the decrease in the expression of the SMAD target genes Id1 and Smad7. These results suggest the existence of another, SMAD-independent pathway of hepcidin gene downregulation.
Highlights
Iron is the oxygen-binding element in hemoglobin and is indispensable for functional erythropoiesis
Original experiments addressing the interaction between iron and EPO on hepcidin expression in mice demonstrated that repeated administration of EPO for four days decreased the elevated expression of Hamp induced by administration of iron-rich diet, as well as the ironinduced liver phosphorylated SMADs (pSMADs) protein content [43]
In agreement with the observed effect of EPO on iron-induced Hamp mRNA content (Fig 1A, column Fe versus FeE), the immunoblot presented in Fig 1E clearly demonstrates that EPO significantly downregulates the high amount of pSMADs induced by a single injection of iron
Summary
Iron is the oxygen-binding element in hemoglobin and is indispensable for functional erythropoiesis. The bone morphogenetic proteins (BMPs) bind, at the extracellular side of the hepatocyte plasma membrane, to heteromeric receptor complexes composed of type I receptors ALK2 and ALK3 and type II receptors BMPR2 and ACTR2A [9, 10]. This binding increases phosphorylation of the receptor-regulated SMAD1, SMAD5 and SMAD9 proteins inside the hepatocyte. The physiological role of TFR2 is puzzling, as several reports describe its expression and function in erythroid cells [15, 16, 17], in addition to its well-established presence on the hepatocyte plasma membrane
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