Research Article: Effect of stimulated erythropoiesis on liver SMAD signaling pathway in iron-overloaded and iron-deficient mice

Date Published: April 8, 2019

Publisher: Public Library of Science

Author(s): Jana Frýdlová, Daniel W. Rogalsky, Jaroslav Truksa, Emanuel Nečas, Martin Vokurka, Jan Krijt, Kostas Pantopoulos.


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.

Partial Text

Iron is the oxygen-binding element in hemoglobin and is therefore indispensable for functional erythropoiesis. In contrast to other transition metals such as copper, molybdenum or cobalt, iron can not be actively excreted from the body, and its homeostasis is regulated only at the level of absorption of dietary iron in the duodenum [1]. The rate limiting process of iron absorption is the transfer of iron from the enterocyte into the bloodstream, which is mediated by the transmembrane iron exporter ferroportin. Ferroportin protein content at the enterocyte basolateral membrane is determined mainly by the circulating levels of the hepatocyte-derived peptide hepcidin, encoded by the Hamp gene [2,3,4].

Hepcidin, the master regulator of systemic iron metabolism, is known to respond to two major stimuli: Iron overload increases Hamp gene expression, whereas accelerated erythropoiesis decreases Hamp expression. According to current concepts, the effect of iron is mediated by increased synthesis of the BMP6 protein, whereas the effect of EPO is partly mediated by increased synthesis of erythroferrone [23, 24, 44], and partly by the EPO-induced redistribution of iron [19, 20]. The molecular basis of the crosstalk between the iron-induced and EPO-induced pathways was first investigated in 2009, when Huang et al. demonstrated that repeated doses of EPO, administered over four days, can inhibit the increase of Hamp mRNA induced by dietary iron, and that this effect correlates with the liver content of phosphorylated SMADs [43]. During further studies on the interplay of the two stimuli, we have reported that, vice versa, a high dose of iron can to a very significant extent prevent the downregulation of Hamp expression elicited by repeated doses of EPO [29]. Thus, in the four-day experimental setting, both stimuli are able to partially antagonize the effect of the other. After the discovery of erythroferrone it was shown that iron-pretreatment blunts the EPO-induced downregulation of Hamp expression in a short-term (15 h) experiment [30], but, in contrast to prolonged EPO administration, EPO in this short-term setting did not attenuate the elevated Hamp expression induced by iron. Based on these observations, it has been postulated that erythroferrone can not efficiently downregulate Hamp expression when the BMP/SMAD pathway is hyperactivated [30, 44]. However, results presented in this study indicate that, in agreement with the original data by Huang et al [43], EPO administration can to some extent decrease the iron-induced increase in phosphorylated SMAD proteins, as well as the iron-induced Hamp and Id1 expression. These data can be interpreted as an in vivo support for the very recent proposal that erythroferrone attenuates signaling through the BMP/SMAD pathway by binding of the BMP6 protein [27]. As can be seen in Fig 1C, liver Bmp6 expression is induced to the same extent both in the iron-treated group and the iron plus EPO-treated group, but Hamp mRNA content, Id1 mRNA content and pSMAD protein content in the iron plus EPO-treated group are significantly reduced (Fig 1A, 1B and 1D, columns Fe versus FeE). The recent hypothesis proposed by Arezes et al. [27], namely that the activity of the BMP/SMAD signaling pathway is blunted by inactivation of the secreted BMP6 protein by circulating erythroferrone, is in agreement with these results.

In conclusion, the study demonstrates that prolonged administration of EPO to C57BL/6 mice decreases the iron-induced hepcidin expression, indicating that accelerated erythropoiesis can downregulate BMP/SMAD signaling even if the BMP/SMAD pathway is hyperactivated, and indirectly supporting the concept that erythroferrone can bind and inactivate the BMP6 protein. It provides new data on the induction of splenic ERFE and TFR2 by EPO, suggesting that the induced levels of these proteins are not further posttranscriptionally regulated by iron overload, but that the EPO-induced splenic TFR2 protein levels are decreased by iron deficiency. It further shows that iron deficiency and EPO administration have an additive effect on the downregulation of liver Hamp expression. Finally, in experiments examining the combined effect of EPO-treatment and iron deficiency, the discrepancy between the limited decrease of liver pSMAD content and Id1 mRNA content on the one hand and the dramatic decrease in Hamp mRNA content on the other strongly suggest the existence of another, SMAD-independent pathway of Hamp gene downregulation.