We used four mouse models of hereditary hemochromatosis, Hfe−/−, Tfr2Y245X/Y245X, Hjv−/−, and Bmp6−/−, in order to examine the role of these proteins in hepcidin regulation by acute and chronic iron loading in vivo. Although Tfr2Y245X/Y245X is not a null mutant, truncated TfR2 is not detectable on the cell membrane both because of nonsense-mediated mRNA decay and defective subcellular transport of the truncated protein.18 All four models were reported to have reduced hepcidin mRNA.7-10, 15, 19 Importantly, to accurately assess hepcidin response to iron loading in mutant mice, we iron-depleted the mutant mice by phlebotomy so that the liver iron content in iron-depleted state was similar to that observed in WT mice.
Acute and Chronic Iron Loading.
We found that chronic iron loading of WT and mutant mice did not raise serum iron more than 1 day iron-loading (Fig. 4). The iron stores in the liver of the mutant mice, however, substantially increased with chronic loading compared with 1-day challenge (Fig. 4). The chronic increase in iron stores was greatest in Hjv and Bmp6 mice, intermediate in Tfr2 mutant mice, and least in Hfe mice. The order of severity of iron loading is the same as reported in the human mutations involving HJV, TfR2, and HFE. On a standard iron diet, WT mice modestly increased their liver iron content compared with 1-day iron loading (Fig. 4, Supporting Information Table 1).
Hepcidin Response to Acute Iron Loading.
After 1-day iron challenge, Tfr2 and Hjv mice did not increase their hepcidin expression, Bmp6 mice increased it minimally, and Hfe mice showed a significant but still blunted response. The result indicates that all four proteins participate in hepcidin regulation by acute iron changes (presumably by holo-Tf concentrations), but that TfR2, HJV, and BMP6 are required for this effect. Interestingly, not only serum iron but also the liver iron stores of WT and mutant mice increased significantly within 1 day of iron challenge, due to the large food consumption by mice relative to their body mass. To confirm that serum iron is a sufficient signal regulating hepcidin expression, we tested hepcidin responsiveness to a holo-Tf injection that contained much smaller amount of iron (14 μg) than was loaded into the liver by a 1-day diet (>100 μg). Hepcidin increased by ≈100-fold within 6 hours compared with solvent controls, and reached maximal hepcidin expression seen in the C57BL/6 mice, indicating that holo-Tf alone can modulate hepcidin mRNA levels.
Hepcidin Response to Chronic Iron Loading.
Hepcidin expression after chronic iron loading of Hfe and Tfr2 mice reached much higher levels than after acute loading and became similar to those of iron-loaded WT mice (Figs. 3 and 5). Although Hjv and Bmp6 mutant mice accumulated the most iron after chronic loading with standard chow, their hepcidin expression rose only modestly and remained severely depressed when assessed relative to their total liver iron load (Fig. 5). The BMP6-deficient iron phenotype may be partially ameliorated if other BMPs associate with HJV to mediate signaling and increase hepcidin expression in response to iron loading. Current models of hepcidin regulation have focused on holo-Tf concentration as the key regulator of hepcidin, through the incompletely defined pathway centered on TfR1 and TfR2 as the likely holo-Tf sensors, interacting with HFE, HJV, and BMP6/BMPR. Increased hepcidin expression after chronic compared with acute iron loading suggests that hepcidin is regulated not only by plasma iron, which is similar after acute and chronic iron loading, but also by intracellular iron stores, which are much higher after chronic than acute iron loading. Based on our findings, the pathway involved in hepcidin regulation by intracellular iron is mainly dependent on HJV and BMP6, with little if any nonredundant contribution from TfR2 or HFE. The greater severity of iron overload in the combined TfR2/HFE deficiency compared with the deficiency of either alone indicates that the two molecules may partially compensate for each other, both in mice and in humans.20, 21 Notably, both Hjv and Bmp6 mice significantly increased hepcidin with chronic iron loading, albeit less than WT strains. Although the loss of either molecule may preserve sufficient BMP receptor activity to regulate hepcidin inefficiently, there is also evidence that alternative pathways are involved in iron regulation. Holo-Tf was reported to activate the extracellular signal-regulated kinase/mitogen-activated protein kinase pathway,6 and it is possible that chronic stimulation of this pathway in Hjv and Bmp6 mice results in hepcidin increase, unrelated to the rise in hepatic iron stores. Alternatively, another as yet unidentified pathway may play a role in sensing intracellular iron and signaling to hepcidin. We did not detect additional hepcidin suppression in diet iron-depleted C57BL/6 mice when they were subjected to phlebotomy (Fig. 2) or any dependence of hepcidin on the severity of postphlebotomy anemia in Hjv and Bmp6 mice (Supporting Information Fig. 3). Nevertheless, our studies cannot exclude that anemia-related erythropoietic factors contribute to the relative hepcidin suppression after phlebotomy and conversely, that the reversal of anemia contributes to the rise of hepcidin after iron loading.
Regulation of Hepcidin by Serum Iron Versus Liver Iron.
Linear multivariate analysis of the dependence of hepcidin mRNA on serum iron versus liver iron revealed that in WT strains, both serum and liver iron contributed to hepcidin regulation. In mutants, only liver iron contributed to hepcidin regulation, and the effect of serum iron was lost. Thus, liver and serum iron may independently regulate hepcidin expression. However, we cannot exclude the possibility that the effects of serum iron are mediated by hepatocyte loading through potential nonlinear effects. For example, at low liver iron concentrations, changes in holo-Tf levels could cause small changes in liver iron content, to which hepcidin mRNA transcription could be highly sensitive. Nevertheless, strong support for the independent regulation of hepcidin by the extracellular effects of serum iron (probably sensed as holo-Tf) is provided by the low hepcidin levels in hypotransferrinemic humans and mice,22, 23 despite hepatic iron overload. Although some of hepcidin decrease could be attributed to the suppressive effects of ineffective erythropoiesis in hypotransferrinemic mice, hepcidin remained low even when the potential effect of erythropoiesis was negated by erythrocyte transfusion or cytotoxic therapy.
BMP6 Responses to Iron Loading.
The expression of Bmp6 mRNA is regulated by iron,24 but the mechanism is not known. The continued rise of Bmp6 mRNA during chronic iron loading parallels the rise in hepatic iron content (Fig. 6), suggesting that BMP6 production is predominantly responsive to intracellular iron stores. However, chronic iron loading of Bmp6 mice resulted in a 270-fold increase in hepcidin mRNA, indicating that BMP6 is not the sole mediator of the intracellular iron signal. Interestingly, when compared with the matching WT strain, hepcidin response to chronic iron loading in Hjv mice appears even more impaired than in Bmp6 mice, despite the significant increase in Bmp6 expression in chronically iron-loaded Hjv mice. This supports the concept that HJV acts as an essential potentiator or coreceptor for BMP6 signaling, but that BMP6 may not be the only factor regulating hepcidin via HJV. Intracellular iron could also signal by altering the cell surface localization of HJV, as indicated by the ability of ferric ammonium citrate or holo-Tf to decrease the release of soluble HJV in vitro.25 Because soluble HJV suppressed hepcidin, but membrane HJV stimulated it,25 this effect of iron would be expected to stimulate hepcidin production. How iron affects the release of soluble HJV is unknown, but the regulation may involve neogenin, a receptor for repulsive guidance molecules,26 or furin, a protease that cleaves HJV.27
In conclusion, our comparative studies of four hereditary hemochromatosis mouse models point to at least two distinct pathways of hepcidin regulation by iron, one sensing extracellular holo-Tf concentrations and the other sensing intracellular iron, presumably in hepatocytes. It is possible that intracellular iron, partially signaling through the HJV/BMP6 pathway, sets basal hepcidin expression, with membrane-bound HJV functioning to heighten the sensitivity of BMP receptors to BMP6 and possibly other ligands. Holo-Tf concentrations signaling through TfR2, HFE, and HJV further increase the sensitivity of BMP receptors to the BMP ligands through as yet unknown mechanisms. It is also possible that TfR2/HFE activate a parallel pathway to increase hepcidin expression. Although not directly addressed in this study, the involvement of nonhepatocyte cell types in the regulation of hepatocyte hepcidin synthesis remains to be explored and could contribute to iron homeostasis.