Heart Ferroportin Protein Content Is Regulated by Heart Iron Concentration and Systemic Hepcidin Expression.

The purpose of the study was to investigate the expression of ferroportin protein following treatments that affect systemic hepcidin. Administration of erythropoietin to C57BL/6J mice decreased systemic hepcidin expression; it also increased heart ferroportin protein content, determined by immunoblot in the membrane fraction, to approximately 200% of control values. This increase in heart ferroportin protein is very probably caused by a decrease in systemic hepcidin expression, in accordance with the classical regulation of ferroportin by hepcidin. However, the control of heart ferroportin protein by systemic hepcidin could apparently be overridden by changes in heart non-heme iron content since injection of ferric carboxymaltose to mice at 300 mg Fe/kg resulted in an increase in liver hepcidin expression, heart non-heme iron content, and also a threefold increase in heart ferroportin protein content. In a separate experiment, feeding an iron-deficient diet to young Wistar rats dramatically decreased liver hepcidin expression, while heart non-heme iron content and heart ferroportin protein content decreased to 50% of controls. It is, therefore, suggested that heart ferroportin protein is regulated primarily by the iron regulatory protein/iron-responsive element system and that the regulation of heart ferroportin by the hepcidin-ferroportin axis plays a secondary role.

Liver HFE protein content is posttranscriptionally decreased in iron-deficient mice and rats.

Although the relationship between hereditary hemochromatosis and mutations in the HFE gene was discovered more than 20 years ago, information on the in vivo regulation of HFE protein expression is still limited. The purpose of the study was to determine the response of liver HFE protein content to iron deficiency in mice and rats by immunoblotting. Attempts to visualize the HFE protein in whole liver homogenates were unsuccessful; however, HFE could be detected in liver microsomes or in plasma membrane-enriched fractions. Five-week-old male C57BL/6 mice fed an iron-deficient diet for 4 wk presented with a significant decrease in liver iron content and liver Hamp expression, as well as with a significant decrease in liver HFE protein content. Rats fed an iron-deficient diet for 4 wk also displayed significant decrease in liver Hamp expression and liver HFE protein content. These results suggest that the downregulation of HFE-dependent signaling may contribute to decreased Hamp gene expression in states of prolonged iron deficiency. It has recently been proposed that HFE protein could be a potential target of matriptase-2, a hepatocyte protease mutated in iron-refractory iron deficiency anemia. However, immunoblot analysis of HFE protein in the livers from Tmprss6-mutated mask mice did not show evidence of matriptase-2-dependent HFE protein cleavage. In addition, no indication of HFE protein cleavage was seen in iron-deficient rats, whereas the full-length matriptase-2 protein content in the same animals was significantly increased. These results suggest that HFE is probably not a major physiological target of matriptase-2. NEW & NOTEWORTHY Feeding of iron-deficient diet for 4 wk decreased liver HFE protein content in both mice and rats, suggesting that decreased HFE-dependent signaling may contribute to hepcidin downregulation in iron deficiency. There was no difference in HFE protein band appearance between matriptase-2-mutated mask mice and wild-type mice, indicating that HFE is probably not a major physiological substrate of matriptase-2-mediated protease activity in vivo.

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

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.