Hereditary hemochromatosis (HH) is a genetically heterogeneous disorder characterized by elevated iron absorption from the diet, with consequent iron overload and tissue injury. Adult-onset forms of HH are caused by mutations in the HFE gene and in the gene for transferrin receptor 2 (TFR2). Human patients and mouse models of TFR2 and HFE-related HH show inappropriately low expression of hepcidin, the central regulator of iron metabolism. However, although these genes have been discovered far more than a decade ago, the mechanisms by which HFE and TFR2 influence hepcidin expression remain unclear.
The bone morphogenetic protein BMP6 plays a key role in the regulation of hepcidin expression. BMP6 binds to type I (ALK3) and type II serine threonine kinase receptors, and to the coreceptor hemojuvelin (HJV), which phosphorylates intracellular SMAD proteins. Phosphorylated SMADs then bind to SMAD4 and translocate to the nucleus to induce the transcription of hepcidin. Inactivation of Bmp6 or Hjv in mice leads to considerably reduced hepcidin production and severe hepatic iron overload. However, there are major differences in hepcidin expression and extrahepatic tissue iron loading between Bmp6 or Hjv KO males and females, due to the suppressive effect of testosterone on hepcidin in males. In contrast to males, Bmp6-/- and Hjv-/-females still produce some hepcidin and do not massively accumulate iron in the pancreas, heart, or kidneys.
The goal of this study was to investigate the role of Hfe and Tfr2 in the residual hepcidin production observed in the absence of Bmp6 in females. We used Bmp6-/-, Tfr2-/-, and B2m-/- mice to generate wild-type, single KO (Bmp6-/-, Tfr2-/-, or B2m-/-) and double KO (Tfr2-/- and Bmp6-/-, or B2m-/- and Bmp6-/-) mice, and we assessed Smad5 phosphorylation, hepcidin expression, and the sites of iron accumulation in the different groups of mice. Notably, B2m-/- mice develop spontaneously hepatic iron overload with a distribution similar to that seen in the liver of Hfe-/-mice and the lack of CD8+ lymphocytes and the absence of classical class I molecules in these mice are not responsible for their iron phenotype.
Interestingly, the lack of functional Hfe or the lack of Tfr2 in Bmp6-/- females leads to a very similar phenotype that is much more severe than the single impairment of Bmp6, with massive iron loading in extrahepatic tissues, most notably the exocrine pancreas, the heart, and the kidney. Hepcidin mRNA and pSmad levels in the two categories of double KO females were much more strongly downregulated than in single Bmp6-/- females and, in contrast to Bmp6-/-females, no protein was detectable by ELISA in the double KO mice.
Our findings clearly demonstrate that Hfe and Tfr2 regulate hepcidin production independently of Bmp6. The symmetrical phenotype of double Bmp6 and Tfr2, or double Bmp6 and Hfe KO mice suggests that Hfe and Tfr2 could participate in the same signaling complex affecting pSmad levels, even if they do not physically interact. Two complexes, one made of ALK3 and HJV, and the other made of ALK2, ALK3, HFE, and TFR2, very likely affect pSMAD levels independently of each other. Signaling through the second complex occurs in the absence of BMP6 and is most probably initiated by another BMP, for instance BMP2 or BMP4 that are also expressed in the liver and, although not regulated by iron, are capable of stimulating hepcidin expression in hepatocytes. Regulation of hepcidin expression by these two complexes would explain why, whereas Alk3, double Bmp6/Hfe, and double Bmp6/Tfr2 deficient females present with iron overload in extrahepatic tissues as do hepcidin KO mice, Bmp6 and Hjv single KO females present with early-onset iron overload only in the liver, similarly to patients with juvenile HH, and mice KO for Alk2, Hfe, or Tfr2 have the least severe phenotype, comparable with that of patients with adult-onset HH. Notably, TFR2 was also shown to localize in lipid rafts where it promotes MAPK activation. Thus, in addition to its role in the complex affecting pSMAD levels, TFR2 could also regulate hepcidin through a parallel pathway involving ERK1/2 signaling. This would explain the more severe phenotype of mice with combined deletion of Hfe and Tfr2 and the fact that, whereas Tfr2-/- mice do not respond to acute iron loading, Hfe-/-mice still have a significant, although blunted hepcidin response.
This work was funded in part by FRM (DEQ2000326528) and ANR (ANR-13-BSV3-0015-01).
No relevant conflicts of interest to declare.
Asterisk with author names denotes non-ASH members.