Contribution of Hfe expression in macrophages to the regulation of hepatic hepcidin levels and iron loading

Abstract

Hereditary hemochromatosis (HH), an iron overload disease associated with mutations in the HFE gene, is characterized by increased intestinal iron absorption and consequent deposition of excess iron, primarily in the liver. Patients with HH and Hfe-deficient (Hfe ؊/؊ ) mice manifest inappropriate expression of the iron absorption regulator hepcidin, a peptide hormone produced by the liver in response to iron loading. In this study, we investigated the contribution of Hfe expression in macrophages to the regulation of liver hepcidin levels and iron loading. We used bone marrow transplantation to generate wild-type (wt) and Hfe ؊/؊ mice chimeric for macrophage Hfe gene expression. Reconstitution of Hfe-deficient mice with wt bone marrow resulted in augmented capacity of the spleen to store iron and in significantly decreased liver iron loading, accompanied by a significant increase of hepatic hepcidin mRNA levels. Conversely, wt mice reconstituted with Hfedeficient bone marrow had a diminished capacity to store iron in the spleen but no significant alterations of liver iron stores or hepcidin mRNA levels. Our results suggest that macrophage Hfe participates in the regulation of splenic and liver iron concentrations and liver hepcidin expression. ( IntroductionHereditary hemochromatosis (HH) type 1, an autosomal recessive disease of iron overload, is one of the most common inherited disorders. It is characterized by failure in the regulation of duodenal iron absorption, leading to iron overloading that can eventually impair organ systems and cause cirrhosis, diabetes, and cardiomyopathy. 1 HH is caused by mutations in the HFE gene encoding a major histocompatibility complex (MHC) class 1-like protein that requires ␤2-microglobulin (B2m) for cell surface expression. 2 A link between HFE and cellular iron metabolism is suggested by the observation that wild-type (wt) HFE-␤ 2 m molecules form a stable complex with transferrin receptor 1 (TfR1). 3 Most patients with HH are homozygous for a missense mutation in the HFE gene that results in cysteine-to-tyrosine substitution at amino acid 282 of HFE protein (C282Y). 2 The mutation disrupts a critical disulfide bond in the ␣ 3 domain of HFE protein and abrogates binding of the mutant HFE protein to B2m, leading to impaired HFE protein intracellular trafficking, incorporation into the cell membrane, and association with TfR1. 3 Studies in mice with targeted inactivation of the Hfe or B2m gene have confirmed the critical role of HFE-B2m complexes in iron metabolism. [4][5][6] As do humans with HH, Hfe-deficient mice develop iron overloading with an accumulation of excess iron, primarily in liver parenchymal cells, as opposed to reticuloendothelial (RE) cell storage characteristic of secondary iron overload. Thus, abnormal regulation of iron metabolism in RE cells seems to take place in HH because these cells are relatively iron deficient compared with surrounding parenchymal hepatocytes. 7 Importantly, RE cells-monocytes and tissue macrophages-have a central role in regulat...

Repression of Repulsive Guidance Molecule C during Inflammation Is Independent of Hfe and Involves Tumor Necrosis Factor-α

“…44 The response to LPS seems to be tissue-specific because Rgmc down-regulation is not seen in the skeletal muscle of mice treated with LPS but is evident in the liver. 22 We further show that down-regulation of Rgmc expression also occurs in the heart, where hepcidin is as well expressed, 31 and persists for up to 48 hours in both the liver and heart. This indicates that Rgmc is important not only in the control of iron metabolism at the systemic level, as it impacts intestinal iron absorption through the regulation of hepcidin levels, 22 but also locally, in the heart.…”

“…mRNA levels of Rgmc, ␤-actin, and hepcidin were measured by real-time PCR in a Rotor Gene 3000 real-time DNA detection system (Montreal Biotech Inc., Kirkland, QC, Canada) with the QuantiTect SYBRϩGreen I PCR kit (Qiagen). 31 The following primers were used: ␤-actin 5Ј-TGTTACCAACTGGGACGACA-3Ј and 5Ј-GGTGTTGAA-GGTCTCAAA-3Ј; Rgmc 5Ј-AATTTCACACATGCCGTGT-C-3Ј and TCAAAGGCTGCAGGAAGATT-3Ј; hepcidin 5Ј-AGAGCTGCAGCCTTTGCAC-3Ј and 5Ј-GAAGATGCAG-ATGGGGAAGT-3Ј. Expression levels were normalized to the housekeeping gene ␤-actin.…”

The effect of iron status on vascular health

“…It has been widely demonstrated that while hemochromatosis is an iron overload state, the macrophage which is responsible for foam cell formation is relatively spared iron accumulation. 22 Whether this is applicable in the heterozygous individuals is not clear. Numerous epidemiological studies have found an association between markers of increased iron status and the risk of atherosclerotic vascular disease.…”

Anemia upregulates lipocalin 2 in the liver and serum

“…Lcn2, hepcidin, and β-actin mRNA levels were measured by real-time PCR in a Rotor Gene 3000™ Real Time DNA Detection System (Montreal Biotech Inc., Kirkland, QC, Canada), with the QuantiTect SYBRGreen I PCR kit (Qiagen, Mississauga, ON, Canada) [14]. The primers were 5′-CCCATCTCTGCTCACTGTCC-3′ and 5′-TTTTTCTGGACCGCATTG-3′ for Lcn2, 5′-AGAGCTGCAGCCTTTGCAC-3′ and 5′-GAAGATGCAGATGGGGAAGT-3′ for hepcidin, and 5′-TGTTACCAACTGGGACGACA-3′ and 5′-GGTGTTGAAGGTCTCAAA-3′ for β-actin.…”