Hepatocyte-targeted HFE and TFR2 control hepcidin expression in mice

Abstract

Hereditary hemochromatosis is caused by mutations in the hereditary hemochromatosis protein (HFE), transferrinreceptor 2 (TfR2), hemojuvelin, hepcidin, or ferroportin genes. Hepcidin is a key iron regulator, which is secreted by the liver, and decreases serum iron levels by causing the down-regulation of the iron transporter, ferroportin. Mutations in either HFE or TfR2 lower hepcidin levels, implying that both HFE and TfR2 are necessary for regulation of hepcidin expression. In this study, we used a recombinant adeno-associated virus, AAV2/8, for hepatocyte-specific expression of either Hfe or Tfr2 in mice. Expression of Hfe in Hfe-null mice both increased Hfe and hepcidin mRNA and lowered hepatic iron and Tf saturation. Expression of Tfr2 in Tfr2-deficient mice had a similar effect, whereas expression of Hfe in Tfr2-deficient mice or of Tfr2 in Hfe-null mice had no effect on liver or serum iron levels. Expression of Hfe in wild-type mice increased hepcidin mRNA and lowered iron levels. In contrast, expression of Tfr2 had no effect on wild-type mice. These findings suggest that Hfe is limiting in formation of the Hfe/Tfr2 complex that regulates hepcidin expression. In addition, these studies show that the use of recombinant AAV vector to deliver genes is a promising approach for studying physiologic consequences of protein complexes. (Blood. 2010;115(16):3374-3381) IntroductionHereditary hemochromatosis (HH) is an autosomal recessive disease of iron metabolism characterized by increased intestinal iron absorption and hepatic iron overload. 1 HH is caused by mutations in genes encoding proteins involved in iron homeostasis, including the HH protein, HFE 2 ; hemojuvelin 3 ; hepcidin 4 ; transferrin-receptor 2 (TfR2) 5 ; and ferroportin. 6 Accumulation of excessive iron results in hepatic cirrhosis, hepatocellular carcinoma, cardiomyopathy, arrhythmias, diabetes, arthritis, and hypogonadotropic hypogonadism. 7 HH type 1, the most common form of HH, is caused by a missense mutation in HFE resulting in a C282Y (numbering system includes the first 23 amino acid signal peptide) substitution and accounts for 85% of HH. 2 HFE encodes an atypical major histocompatibility complex class I protein. Like the major histocompatibility complex class I proteins, HFE is a membrane protein that consists of a signal sequence, ␣1-␣3 domains followed by a transmembrane domain, and a short cytoplasmic domain. HFE also forms a heterodimeric complex with ␤2-microglobulin. 8 The C282Y mutation in HFE disrupts a disulfide bond in the ␣3 domain, leading to misfolding, lack of association with ␤2-microglobulin, and failure to traffic to the cell surface. 9 The Hfe knockout mouse (Hfe Ϫ/Ϫ ) also develops iron overload, confirming that the HFE-C282Y mutation confers loss of rather than gain of function. 10 Although the importance of HFE in iron regulation is apparent in patients with HH and murine models, 10,11 the underlying mechanism by which HFE regulates iron metabolism is only beginning to be understood.Type 3 HH is caused by a variety o...

The Role of Hepatocyte Hemojuvelin in the Regulation of Bone Morphogenic Protein-6 and Hepcidin Expression in Vivo

“…3A). These results are consistent with previous reports (22,29,30), showing the specificity of AAV2/8 vector to introduce the gene of interest into hepatocytes. Together, our data suggest that AAV2/8 vector targets Hjv into the hepatocytes for expression.…”

“…6C). These observations, along with previous findings that AAV2/8 vector does not cause inflammation in mice and that administration of control vector does not change hepcidin expression (30), ruled out inflammation as a factor. Together, our data support the model that hepatocyte HJV induces hepcidin expression through potentiating BMP6-mediated signaling, rather than by stimulating BMP6 expression.…”

Human iron transporters

“…In this context, the critical involvement of the liver in TfR2 (and HFE) modulation of hepcidin has just been confirmed [13] in mice. Another study [41] examines the role of two isoforms of TfR2 mRNA.…”

CD81 Promotes Both the Degradation of Transferrin Receptor 2 (TfR2) and the Tfr2-mediated Maintenance of Hepcidin Expression

“…qRT-PCR-Total RNA was isolated from Hep3B cells using the RNAeasy RNA isolation kit (Qiagen) and treated with DNase (Roche Applied Science) to remove any contaminating genomic DNA as previously described (14). Oligo(dT) primers and Superscript II reverse transcriptase were used to synthesize cDNA according to the manufacturer's instructions.…”

“…TfR2 is proposed to sense iron levels in the blood and positively regulate the expression of hepcidin. Our previous studies using an adeno-associated virus vector (rAAV2/8) to express Tfr2 in hepatocytes of Tfr2-deficient mice demonstrated that Tfr2 expression increases hepcidin expression and reduces hepatic iron overload in the Tfr2-deficient mice (14), directly suggesting that TfR2 regulates hepcidin expression. However, the mechanism and signaling pathway by which TfR2 regulates hepcidin expression remains to be determined.…”