Erythropoietin regulates intestinal iron absorption in a rat model of chronic renal failure.

Erythropoietin is produced by the kidney and stimulates erythropoiesis; however, in chronic renal disease its levels are reduced and patients develop anemia that is treatable with iron and recombinant hormone. The mechanism by which erythropoietin improves iron homeostasis is still unclear, but it may involve suppression of the iron regulatory peptide hepcidin and/or a direct effect on intestinal iron absorption. To investigate these possibilities, we used the well-established 5/6th nephrectomy rat model of chronic renal failure with or without human recombinant erythropoietin treatment. Monolayers of human intestinal Caco-2 cells were also treated with erythropoietin to measure any direct effects of this hormone on intestinal iron transport. Nephrectomy increased hepatic hepcidin expression and decreased intestinal iron absorption; these effects were restored to levels found in sham-operated rats on erythropoietin treatment of the rats with renal failure. In Caco-2 cells, the addition of erythropoietin significantly increased the expression of apical divalent metal transporter 1 (DMT1) and basolateral ferroportin and, consequently, iron transport across the monolayer. Taken together, our results show that erythropoietin not only exerts a powerful inhibitory action on the expression of hepcidin, thus permitting the release of iron from reticuloendothelial macrophages and intestinal enterocytes, but also acts directly on enterocytes to increase iron absorption.

Transferrin receptor 2 is crucial for iron sensing in human hepatocytes.

Hepcidin expression in vivo is regulated in proportion to iron status (i.e., increased by iron loading and decreased in iron deficiency). However, in vitro studies with hepatoma cell lines often show an inverse relationship between iron status and hepcidin expression. Here, we investigated possible molecular mechanisms responsible for the differences in iron sensing between hepatoma cell lines and human primary hepatocytes. RNA was collected from primary human hepatocytes, and HepG2 and HuH7 hepatoma cells were treated with either transferrin-bound and non-transferrin-bound iron. Expression of hepcidin, transferrin receptor 2, HFE, and hemojuvelin were quantified by real-time PCR. Hepcidin expression was increased in primary human hepatocytes following 24-h exposure to holoferric transferrin. In contrast, hepcidin mRNA levels in hepatoma cells were decreased by transferrin. Hepcidin expression was positively correlated with transferrin receptor 2 mRNA levels in primary human hepatocytes. Compared with primary hepatocytes, transferrin receptor 2 expression was significantly lower in hepatoma cell lines; furthermore, there was no correlation between transferrin receptor 2 and hepcidin mRNA levels in either HepG2 or HuH7 cells. Taken together our data suggest that transferrin receptor 2 is a likely candidate to explain the differences in iron sensing between hepatoma cell lines and primary human hepatocytes.

Biochemical and spectroscopic studies of human melanotransferrin (MTf): electron-paramagnetic resonance evidence for a difference between the iron-binding site of MTf and other transferrins.

Melanotransferrin (MTf) is a member of the transferrin (Tf) family of iron (Fe)-binding proteins that was first identified as a cell-surface marker of melanoma. Although MTf has a high-affinity Fe-binding site that is practically identical to that of serum Tf, the protein does not play an essential role in Fe homeostasis and its precise molecular function remains unclear. A Zn(II)-binding motif, distinct from the Fe-binding site, has been proposed in human MTf based on computer modelling studies. However, little is known concerning the interaction of its proposed binding site(s) with metals and the consequences in terms of MTf conformation. For the first time, biochemical and spectroscopic techniques have been used in this study to characterise metal ion-binding to recombinant MTf. Initially, the binding of Fe to MTf was examined using 6M urea gel electrophoresis. Although four different iron-loaded forms were observed with serum Tf, only two forms were found with MTf, the apo-form and the N-monoferric holo-protein, suggesting a single high-affinity site. The presence of a single Fe(III)-binding site was also supported by EPR results which indicated that the Fe(III)-binding characteristics of MTf were unique, but somewhat comparable to the N-lobes of human serum Tf and chicken ovo-Tf. Circular dichroism (CD) analysis indicated that, as for Tf, no changes in secondary structure could be observed upon Fe(III)-binding. The ability of MTf to bind Zn(II) was also investigated using CD which demonstrated that the single high-affinity Fe-binding site was distinct from a potential Zn(II)-binding site.

Identification of an iron-hepcidin complex.

Following its identification as a liver-expressed antimicrobial peptide, the hepcidin peptide was later shown to be a key player in iron homoeostasis. It is now proposed to be the 'iron hormone' which, by interacting with the iron transporter ferroportin, prevents further iron import into the circulatory system. This conclusion was reached using the corresponding synthetic peptide, emphasizing the functional importance of the mature 25-mer peptide, but omitting the possible functionality of its maturation. From urine-purified native hepcidin, we recently demonstrated that a proportion of the purified hepcidin had formed iron-hepcidin complexes. This interaction was investigated further by computer modelling and, based on the sequence similarity of hepcidin with metallothionein, a three-dimensional model of hepcidin, containing one atom of iron, was constructed. To characterize these complexes further, the interaction with iron was analysed using different spectroscopic methods. Monoferric hepcidin was identified by MS, as were possibly other complexes containing two and three atoms of iron respectively, although these were present only in minor amounts. UV/visible absorbance and CD studies identified the iron-binding events which were facilitated at a physiological pH. EPR spectroscopy identified the ferric state of the bound metal, and indicated that the iron-hepcidin complex shares some similarities with the rubredoxin iron-sulfur complex, suggesting the presence of Fe(3+) in a tetrahedral sulfur co-ordination. The potential roles of iron binding for hepcidin are discussed, and we propose either a regulatory function in the maturation of pro-hepcidin into active hepcidin or as the necessary link in the interaction between hepcidin and ferroportin.

Nature of non-transferrin-bound iron: studies on iron citrate complexes and thalassemic sera.

Despite its importance in iron-overload diseases, little is known about the composition of plasma non-transferrin-bound iron (NTBI). Using 30-kDa ultrafiltration, plasma from thalassemic patients consisted of both filterable and non-filterable NTBI, the filterable fraction representing less than 10% NTBI. Low filterability could result from protein binding or NTBI species exceeding 30 kDa. The properties of iron citrate and its interaction with albumin were therefore investigated, as these represent likely NTBI species. Iron permeated 5- or 12-kDa ultrafiltration units completely when complexes were freshly prepared and citrate exceeded iron by tenfold, whereas with 30-kDa ultrafiltration units, permeation approached 100% at all molar ratios. A g = 4.3 electron paramagnetic resonance signal, characteristic of mononuclear iron, was detectable only with iron-to-citrate ratios above 1:100. The ability of both desferrioxamine and 1,2-dimethyl-3-hydroxypyridin-4-one to chelate iron in iron citrate complexes also increased with increasing ratios of citrate to iron. Incremental molar excesses of citrate thus favour the progressive appearance of chelatable lower molecular weight iron oligomers, dimers and ultimately monomers. Filtration of iron citrate in the presence of albumin showed substantial binding to albumin across a wide range of iron-to-citrate ratios and also increased accessibility of iron to chelators, reflecting a shift towards smaller oligomeric species. However, in vitro experiments using immunodepletion or absorption of albumin to Cibacron blue-Sepharose indicate that iron is only loosely bound in iron citrate-albumin complexes and that NTBI is unlikely to be albumin-bound to any significant extent in thalassemic sera.

Structure-function analysis of Arg-Gly-Asp helix motifs in alpha v beta 6 integrin ligands.

Data relating to the structural basis of ligand recognition by integrins are limited. Here we describe the physical requirements for high affinity binding of ligands to alpha v beta6. By combining a series of structural analyses with functional testing, we show that 20-mer peptide ligands, derived from high affinity ligands of alpha v beta6 (foot-and-mouth-disease virus, latency associated peptide), have a common structure comprising an Arg-Gly-Asp motif at the tip of a hairpin turn followed immediately by a C-terminal helix. This arrangement allows two conserved Leu/Ile residues at Asp(+1) and Asp(+4) to be presented on the outside face of the helix enabling a potential hydrophobic interaction with the alpha v beta6 integrin, in addition to the Arg-Gly-Asp interaction. The extent of the helix determines peptide affinity for alpha v beta6 and potency as an alpha v beta6 antagonist. A major role of this C-terminal helix is likely to be the correct positioning of the Asp(+1) and Asp(+4) residues. These data suggest an explanation for several biological functions of alpha v beta6 and provide a structural platform for design of alpha v beta6 antagonists.