Heterologous downregulation of vasopressin type 2 receptor is induced by transferrin.

Vasopressin (VP) binds to the vasopressin type 2 receptor (V2R) to trigger physiological effects including body fluid homeostasis and blood pressure regulation. Signaling is terminated by receptor downregulation involving clathrin-mediated endocytosis and V2R degradation. We report here that both native and epitope-tagged V2R are internalized from the plasma membrane of LLC-PK1 kidney epithelial cells in the presence of another ligand, transferrin (Tf). The presence of iron-saturated Tf (holo-Tf; 4 h) reduced V2R binding sites at the cell surface by up to 33% while iron-free (apo-Tf) had no effect. However, no change in green fluorescent protein-tagged V2R distribution was observed in the presence of bovine serum albumin, atrial natriuretic peptide, or ANG II. Conversely, holo-Tf did not induce the internalization of another G protein-coupled receptor, the parathyroid hormone receptor. In contrast to the effect of VP, Tf did not increase intracellular cAMP or modify aquaporin-2 distribution in these cells, although addition of VP and Tf together augmented VP-induced V2R internalization. Tf receptor coimmunoprecipitated with V2R, suggesting that they interact closely, which may explain the additive effect of VP and Tf on V2R endocytosis. Furthermore, Tf-induced V2R internalization was abolished in cells expressing a dominant negative dynamin (K44A) mutant, indicating the involvement of clathrin-coated pits. We conclude that Tf can induce heterologous downregulation of the V2R and this might desensitize VP target cells without activating downstream V2R signaling events. It also provides new insights into urine-concentrating defects observed in rat models of hemochromatosis.

Nramp1 promotes efficient macrophage recycling of iron following erythrophagocytosis in vivo.

Natural resistance-associated macrophage protein 1 (Nramp1) is a divalent metal transporter expressed exclusively in phagocytic cells. We hypothesized that macrophage Nramp1 may participate in the recycling of iron acquired from phagocytosed senescent erythrocytes. To evaluate the role of Nramp1 in vivo, the iron parameters of WT and KO mice were analyzed after acute and chronic induction of hemolytic anemia. We found that untreated KO mice exhibited greater serum transferrin saturation and splenic iron content with higher duodenal ferroportin (Fpn) and divalent metal transporter 1 (DMT1) expression. Furthermore, hepatocyte iron content and hepcidin mRNA levels were dramatically lower in KO mice, indicating that hepcidin levels can be regulated by low-hepatocyte iron stores despite increased transferrin saturation. After acute treatment with the hemolytic agent phenylhydrazine (Phz), KO mice experienced a significant decrease in transferrin saturation and hematocrit, whereas WT mice were relatively unaffected. After a month-long Phz regimen, KO mice retained markedly increased quantities of iron within the liver and spleen and exhibited more pronounced splenomegaly and reticulocytosis than WT mice. After injection of (59)Fe-labeled heat-damaged reticulocytes, KO animals accumulated erythrophagocytosed (59)Fe within their liver and spleen, whereas WT animals efficiently recycled phagocytosed (59)Fe to the marrow and erythrocytes. These data imply that without Nramp1, iron accumulates within the liver and spleen during erythrophagocytosis and hemolytic anemia, supporting our hypothesis that Nramp1 promotes efficient hemoglobin iron recycling in macrophages. Our observations suggest that mutations in Nramp1 could result in a novel form of human hereditary iron overload.

Bone morphogenetic protein signaling is impaired in an HFE knockout mouse model of hemochromatosis.

BACKGROUND AND AIMS: Mutations in HFE are the most common cause of the iron-overload disorder hereditary hemochromatosis. Levels of the main iron regulatory hormone, hepcidin, are inappropriately low in hereditary hemochromatosis mouse models and patients with HFE mutations, indicating that HFE regulates hepcidin. The bone morphogenetic protein 6 (BMP6)-SMAD signaling pathway is an important endogenous regulator of hepcidin expression. We investigated whether HFE is involved in BMP6-SMAD regulation of hepcidin expression. METHODS: The BMP6-SMAD pathway was examined in Hfe knockout (KO) mice and in wild-type (WT) mice as controls. Mice were placed on diets of varying iron content. Hepcidin induction by BMP6 was examined in primary hepatocytes from Hfe KO mice; data were compared with those of WT mice. RESULTS: Liver levels of Bmp6 messenger RNA (mRNA) were higher in Hfe KO mice; these were appropriate for the increased hepatic levels of iron in these mice, compared with WT mice. However, levels of hepatic phosphorylated Smad 1/5/8 protein (an intracellular mediator of Bmp6 signaling) and Id1 mRNA (a target gene of Bmp6) were inappropriately low for the body iron burden and Bmp6 mRNA levels in Hfe KO, compared with WT mice. BMP6 induction of hepcidin expression was reduced in Hfe KO hepatocytes compared with WT hepatocytes. CONCLUSIONS: HFE is not involved in regulation of BMP6 by iron, but does regulate the downstream signals of BMP6 that are triggered by iron.

BMP6 is a key endogenous regulator of hepcidin expression and iron metabolism.

Juvenile hemochromatosis is an iron-overload disorder caused by mutations in the genes encoding the major iron regulatory hormone hepcidin (HAMP) and hemojuvelin (HFE2). We have previously shown that hemojuvelin is a co-receptor for bone morphogenetic proteins (BMPs) and that BMP signals regulate hepcidin expression and iron metabolism. However, the endogenous BMP regulator(s) of hepcidin in vivo is unknown. Here we show that compared with soluble hemojuvelin (HJV.Fc), the homologous DRAGON.Fc is a more potent inhibitor of BMP2 or BMP4 but a less potent inhibitor of BMP6 in vitro. In vivo, HJV.Fc or a neutralizing antibody to BMP6 inhibits hepcidin expression and increases serum iron, whereas DRAGON.Fc has no effect. Notably, Bmp6-null mice have a phenotype resembling hereditary hemochromatosis, with reduced hepcidin expression and tissue iron overload. Finally, we demonstrate a physical interaction between HJV.Fc and BMP6, and we show that BMP6 increases hepcidin expression and reduces serum iron in mice. These data support a key role for BMP6 as a ligand for hemojuvelin and an endogenous regulator of hepcidin expression and iron metabolism in vivo.

Alcohol metabolism-mediated oxidative stress down-regulates hepcidin transcription and leads to increased duodenal iron transporter expression.

Patients with alcoholic liver disease frequently exhibit iron overload in association with increased hepatic fibrosis. Even moderate alcohol consumption elevates body iron stores; however, the underlying molecular mechanisms are unknown. Hepcidin, a circulatory peptide synthesized in the liver, is a key mediator of iron metabolism. Ethanol metabolism significantly down-regulated both in vitro and in vivo hepcidin mRNA and protein expression. 4-Methylpyrazole, a specific inhibitor of the alcohol-metabolizing enzymes, abolished the effects of ethanol on hepcidin. However, ethanol did not alter the expression of transferrin receptor1 and ferritin or the activation of iron regulatory RNA-binding proteins, IRP1 and IRP2. Mice maintained on 10-20% ethanol for 7 days displayed down-regulation of liver hepcidin expression without changes in liver triglycerides or histology. This was accompanied by elevated duodenal divalent metal transporter1 and ferroportin protein expression. Injection of hepcidin peptide negated the effect of ethanol on duodenal iron transporters. Ethanol down-regulated hepcidin promoter activity and the DNA binding activity of CCAAT/enhancer-binding protein alpha (C/EBPalpha) but not beta. Interestingly, the antioxidants vitamin E and N-acetylcysteine abolished both the alcohol-mediated down-regulation of C/EBPalpha binding activity and hepcidin expression in the liver and the up-regulation of duodenal divalent metal transporter 1. Collectively, these findings indicate that alcohol metabolism-mediated oxidative stress regulates hepcidin transcription via C/EBPalpha, which in turn leads to increased duodenal iron transport.