Sarah J. Wilkins

A rapid decrease in the expression of DMT1 and Dcytb but not Ireg1 or hephaestin explains the mucosal block phenomenon of iron absorption

Background: A large oral dose of iron will reduce the absorption of a subsequent smaller dose of iron in a phenomenon known as mucosal block. Molecular analysis of this process may provide insights into the regulation of intestinal iron absorption. Aims: To determine the effect of an oral bolus of iron on duodenal expression of molecules associated with intestinal iron transport in rats and to relate this to changes in iron absorption. Methods: Rats were given an oral dose of iron and duodenal expression of divalent metal transporter 1 (DMT1), Dcytb, Ireg1, and hephaestin (Hp) was determined using the ribonuclease protection assay, western blotting, and immunofluorescence. Iron absorption was measured using radioactive 59Fe. Results: A decrease in intestinal iron absorption occurred following an oral dose of iron and this was associated with increased enterocyte iron levels, as assessed by iron regulatory protein activity and immunoblotting for ferritin. Reduced absorption was also accompanied by a rapid decrease in expression of the mRNAs encoding the brush border iron transport molecules Dcytb and the iron responsive element (IRE) containing the splice variant of DMT1. No such change was seen in expression of the non-IRE splice variant of DMT1 or the basolateral iron transport molecules Ireg1 and Hp. Similar changes were observed at the protein level. Conclusions: These data indicate that brush border, but not basolateral, iron transport components are regulated locally by enterocyte iron levels and support the hypothesis that systemic stimuli exert their primary effect on basolateral transport molecules.

Delayed hepcidin response explains the lag period in iron absorption following a stimulus to increase erythropoiesis

Introduction: The delay of several days between an erythropoietic stimulus and the subsequent increase in intestinal iron absorption is commonly believed to represent the time required for body signals to programme the immature crypt enterocytes and for these cells to migrate to the villus. Recent data however suggest that signals from the body to alter absorption are mediated by circulating hepcidin and that this peptide exerts its effect on mature villus enterocytes. Methods: We have examined the delay in the absorptive response following stimulated erythropoiesis using phenylhydrazine induced haemolysis and correlated this with expression of hepcidin in the liver and iron transporters in the duodenum. Results: There was a delay of four days following haemolysis before a significant increase in iron absorption was observed. Hepatic hepcidin expression did not decrease until day 3, reaching almost undetectable levels by days 4 and 5. This coincided with the increase in duodenal expression of divalent metal transporter 1, duodenal cytochrome b, and Ireg1. Conclusion: These results suggest that the delayed increase in iron absorption following stimulated erythropoiesis is attributable to a lag in the hepcidin response rather than crypt programming, and are consistent with a direct effect of the hepcidin pathway on mature villus enterocytes.

A genome-wide screen for modifiers of transgene variegation identifies genes with critical roles in development

BackgroundSome years ago we established an N-ethyl-N-nitrosourea screen for modifiers of transgene variegation in the mouse and a preliminary description of the first six mutant lines, named MommeD1-D6, has been published. We have reported the underlying genes in three cases: MommeD1 is a mutation in SMC hinge domain containing 1 (Smchd1), a novel modifier of epigenetic gene silencing; MommeD2 is a mutation in DNA methyltransferase 1 (Dnmt1); and MommeD4 is a mutation in Smarca 5 (Snf2h), a known chromatin remodeler. The identification of Dnmt1 and Smarca5 attest to the effectiveness of the screen design.ResultsWe have now extended the screen and have identified four new modifiers, MommeD7-D10. Here we show that all ten MommeDs link to unique sites in the genome, that homozygosity for the mutations is associated with severe developmental abnormalities and that heterozygosity results in phenotypic abnormalities and reduced reproductive fitness in some cases. In addition, we have now identified the underlying genes for MommeD5 and MommeD10. MommeD5 is a mutation in Hdac1, which encodes histone deacetylase 1, and MommeD10 is a mutation in Baz1b (also known as Williams syndrome transcription factor), which encodes a transcription factor containing a PHD-type zinc finger and a bromodomain. We show that reduction in the level of Baz1b in the mouse results in craniofacial features reminiscent of Williams syndrome.ConclusionsThese results demonstrate the importance of dosage-dependent epigenetic reprogramming in the development of the embryo and the power of the screen to provide mouse models to study this process.

Regulation of systemic iron homeostasis: how the body responds to changes in iron demand

The iron that is required to meet the metabolic needs of cells and tissues is derived from the plasma. Plasma iron in turn reflects the release of iron from various body cells, principally the macrophages of the reticuloendothelial system, and the absorption of dietary iron by the proximal small intestine. This iron donation is highly regulated and the liver-derived peptide hepcidin has emerged as the key modulator of cellular iron export. Following its synthesis and secretion from the liver, circulating hepcidin reduces iron export into the plasma by binding to the iron efflux protein ferroportin1 on the surface of enterocytes, macrophages and other cell types and causing its internalization. The level of hepatic hepcidin expression is influenced by HFE, transferrin receptor 2 and hemojuvelin, and the signal transduction pathway(s) linking these proteins to hepcidin are only beginning to be revealed. Hemojuvelin has recently been shown to signal through the bone morphogenetic protein pathway, ultimately activating receptor SMAD/SMAD4 complexes to alter hepcidin transcription. Circulating differic transferrin has emerged as a possible upstream regulator of the liver-based hepcidin regulatory pathway. In addition to being regulated by body iron requirements, hepcidin expression can be modulated by pro-inflammatory cytokines such as interleukin-6. The continuing analysis of inherited disorders of iron metabolism combined with biochemical analysis of signal transduction pathways is essential to fully define this important regulatory system.

Changes in the expression of intestinal iron transport and hepatic regulatory molecules explain the enhanced iron absorption associated with pregnancy in the rat

Background: Iron absorption increases during pregnancy to cater for the increased iron requirements of the growing fetus. Aims: To investigate the role of the duodenal iron transport molecules and hepatic regulatory molecules in coordinating the changes in iron absorption observed during pregnancy. Methods: Rats at various days of gestation and 24–48 hours post-partum were examined for hepatic expression of hepcidin, transferrin receptors 1 and 2, and HFE (the gene mutated in the most prevalent form of hereditary haemochromatosis), and duodenal expression of divalent metal transporter 1 (DMT1), duodenal cytochrome b (Dcytb), iron regulated mRNA (Ireg1), and hephaestin (Hp) by ribonuclease protection assay, western blotting, and immunohistochemistry. Results: Decreased hepatic non-haem iron and transferrin saturation and increased expression of transferrin receptor 1 in the liver indicated a progressive reduction in maternal body iron stores during pregnancy. Duodenal expression of the iron transport molecules DMT1, Dcytb, and Ireg1 increased during pregnancy, and this corresponded with a reduction in hepcidin, HFE, and transferrin receptor 2 expression in the liver. Expression of all molecules returned towards control values by 24–48 hours post-partum. Conclusions: These data indicate that increased expression of key iron transport molecules is responsible for the elevated iron absorption associated with pregnancy, and implicate hepcidin, HFE, and transferrin receptor 2 in determining how the maternal iron homeostatic machinery responds to the increased iron demands accompanying gestation.

Increased hepcidin expression and hypoferraemia associated with an acute phase response are not affected by inactivation of HFE

The effect of HFE inactivation on iron homeostasis during an acute phase response was investigated in mice. HFE knockout, β2‐microglobulin knockout and C57BL/6J mice were injected with Freunds Complete Adjuvant to induce an acute phase response and hepatic hepcidin expression and serum transferrin saturation was determined 16 h later. Hepcidin mRNA increased in all strains in response to an acute phase stimulus when compared with untreated control animals. Hypoferraemia also occurred in all strains, indicating that both the upregulation of hepcidin and the decrease in transferrin saturation associated with an acute phase response is not dependent on HFE function.

Stimulated erythropoiesis with secondary iron loading leads to a decrease in hepcidin despite an increase in bone morphogenetic protein 6 expression

The BMP/SMAD signalling pathway plays an important role in iron homeostasis, regulating hepcidin expression in response to body iron levels. However, the role of this pathway in the reduction in hepcidin associated with increased erythropoiesis (and secondary iron loading) is unclear. To investigate this, we established a mouse model of chronic stimulated erythropoiesis with secondary iron loading using the haemolytic agent phenylhydrazine. We then examined the expression of components of the BMP6/SMAD signalling pathway in these animals. We also examined this pathway in the Hbbth3/+ mouse, a model of the iron loading anaemia β‐thalassaemia intermedia. Increasing doses of phenylhydrazine led to a progressive increase in both liver iron levels and Bmp6 mRNA expression, but, in contrast, hepatic Hamp expression declined. The increase in Bmp6 expression was not associated with a corresponding change in the phosphorylation of hepatic SMAD1/5/8, indicating that stimulated erythropoiesis decreases the ability of BMP6 to alter SMAD phosphorylation. Increased erythropoiesis also reduces the capacity of phosphorylated SMAD (pSMAD) to induce hepcidin, as Hamp levels declined despite no changes in pSMAD1/5/8. Similar results were seen in Hbbth3/+ mice. Thus the erythroid signal probably affects some components of BMP/SMAD signalling, but also may exert some independent effects.

The multicopper ferroxidase hephaestin enhances intestinal iron absorption in mice

Hephaestin is a vertebrate multicopper ferroxidase important for the transfer of dietary iron from intestinal cells to the blood. Hephaestin is mutated in the sex-linked anemia mouse, resulting in iron deficiency. However, sex-linked anemia mice still retain some hephaestin ferroxidase activity. They survive, breed, and their anemia improves with age. To gain a better understanding of the role of hephaestin in iron homeostasis, we used the Cre-lox system to generate knockout mouse models with whole body or intestine-specific (Villin promoter) ablation of hephaestin. Both types of mice were viable, indicating that hephaestin is not essential and that other mechanisms, multicopper ferroxidase-dependent or not, must compensate for hephaestin deficiency. The knockout strains, however, both developed a microcytic, hypochromic anemia, suggesting severe iron deficiency and confirming that hephaestin plays an important role in body iron acquisition. Consistent with this, the knockout mice accumulated iron in duodenal enterocytes and had reduced intestinal iron absorption. In addition, the similarities of the phenotypes of the whole body and intestine-specific hephaestin knockout mice clarify the important role of hephaestin specifically in intestinal enterocytes in maintaining whole body iron homeostasis. These mouse models will serve as valuable tools to study the role of hephaestin and associated proteins in iron transport in the small intestine and other tissues.

Severe iron deficiency blunts the response of the iron regulatory gene Hamp and pro-inflammatory cytokines to lipopolysaccharide

Background Expression of the key iron regulatory hormone hepcidin is increased by some stimuli (iron loading, inflammation) but decreased by others (increased erythropoiesis, iron deficiency). We investigated the response of hepcidin to increased erythropoiesis and iron deficiency in the presence of an acute inflammation to assess the relative strengths of these stimuli. Design and Methods Sprague-Dawley rats were maintained on control or iron-deficient diets and treated with lipopolysaccharide to induce inflammation or phenylhydrazine to stimulate erythropoiesis. The levels of Hamp, IL-6 and α2m mRNA were determined by qualitative real-time polymerase chain reaction and those of serum interleukin-6 and tumor necrosis factor-α were measured by enzyme-linked immunosorbent assay. Cultured RAW264.7 and HuH7 cells were used in associated studies. Results The increase in hepatic hepcidin levels induced by lipopolysaccharide was not affected by phenylhydrazine treatment but was blunted by iron deficiency. Lipopolysaccharide-treated iron-deficient animals also showed lower liver α2m mRNA and reduced serum interleukin-6 and tumor necrosis factor-α, suggesting a more generalized effect of iron deficiency. Similarly, RAW 264.7 cells treated with iron chelators and then stimulated with lipopolysaccharide showed lower IL-6 mRNA than cells treated with lipopolysaccharide alone. Huh7 cells treated with an iron chelator showed a blunted hepcidin response to interleukin-6, suggesting that the response of hepatic parenchymal cells to inflammatory cytokines may also be iron-dependent. Conclusions In any one physiological situation, net hepcidin levels are determined by the relative strengths of competing stimuli. The ability of severe iron deficiency to blunt the response to lipopolysaccharide of both hepcidin and other markers of inflammation suggests that adequate iron levels are necessary for a full acute phase response.

Postnatal growth restriction and gene expression changes in a mouse model of fetal alcohol syndrome

Growth restriction, craniofacial dysmorphology, and central nervous system defects are the main diagnostic features of fetal alcohol syndrome. Studies in humans and mice have reported that the growth restriction can be prenatal or postnatal, but the underlying mechanisms remain unknown.We recently described a mouse model of moderate gestational ethanol exposure that produces measurable phenotypes in line with fetal alcohol syndrome (e.g., craniofacial changes and growth restriction in adolescent mice). In this study, we characterize in detail the growth restriction phenotype by measuring body weight at gestational day 16.5, cross-fostering from birth to weaning, and by extending our observations into adulthood. Furthermore, in an attempt to unravel the molecular events contributing to the growth phenotype, we have compared gene expression patterns in the liver and kidney of nonfostered, ethanol-exposed and control mice at postnatal day 28.We find that the ethanol-induced growth phenotype is not detectable prior to birth, but is present at weaning, even in mice that have been cross-fostered to unexposed dams. This finding suggests a postnatal growth restriction phenotype that is not due to deficient postpartum care by dams that drank ethanol, but rather a physiologic result of ethanol exposure in utero. We also find that, despite some catch-up growth after 5 weeks of age, the effect extends into adulthood, which is consistent with longitudinal studies in humans.Genome-wide gene expression analysis revealed interesting ethanol-induced changes in the liver, including genes involved in the metabolism of exogenous and endogenous compounds, iron homeostasis, and lipid metabolism.