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How is iron transport across the placenta regulated?

Reference	BB/D521322/1
Principal Investigator / Supervisor	Professor Surjit Srai
Co-Investigators / Co-Supervisors
Institution	University College London
Department	Structural Molecular Biology
Funding type	Research
Value (£)	410,632
Status	Completed
Type	Research Grant
Start date	01/11/2005
End date	28/02/2009
Duration	40 months

Abstract

Although we understand some aspects of the mechanism of iron transport across the placenta, we still do not know the signalling molecules involved in this regulation. In the duodenum, two proteins, HFE and hepcidin, have received particular attention. The aim of this study is to determine whether they have a similar role in the regulation of iron uptake and efflux across the placenta into the foetal circulation. We will examine separately the importance of maternal liver HFE and placental HFE in regulation of Fe delivery. Whether maternal hepcidin plays a role will also be tested. The mechanism of interaction will be studied using an in vitro model for placenta, the BeWo cell, which we and others have shown is a good model for Fe metabolism in placenta. The data will also indicate whether maternal hepcidin could be used as a biomarker of maternal Fe status. This is particularly important since we know that the present indicators are neither sensitive nor accurate in giving maternal Fe stores or supply. Hypothesis: Hepcidin and HFE are involved in the modulation of iron uptake and/or efflux across the placenta by regulating the expression of genes involved in iron transport across the syncytiotrophoblast. To examine the role of maternal HFE, we will mate HFE(-/-) or wild type (+/+) females with HFE(-/+) males. The foetuses thus generated will be wild type heterozygote, or homozygous mutant. We will measure Fe levels in the foetal liver, which will give a clear indication whether transport is compromised. If maternal HFE is most important, all the offspring of an HFE -/- dam will have low Fe, irrespective of their own phenotype. If the foetal genotype is most important, the response would be normal, intermediate or low, depending on the genotype. We will be able to examine how the effect is mediated by measuring expression of the proteins involved in Fe transfer. On the uptake side, these are transferrin receptor (TfR) and DMT1. On the efflux pathway, these are IREG1and eleutherin. In rat experiments, we have shown that TfR, DMT1 and eleutherin are increased in Fe deficiency, while IREG1 is not changed. To test whether maternal hepcidin plays a role in this process, we will feed wild type mice an Fe deficient diet. This reduces hepcidin levels in maternal serum and we will return levels to approximately normal in an experimental group by injecting daily with hepcidin (s.c.). As control, we will inject a scrambled version of the same amino acids. Depending on the function of the peptide, the Fe levels will be restored to normal, or not. However, these experiments cannot determine whether there is a direct or an indirect role for hepcidin. To test these possibilities, we will use BeWo cells. These are a placental cell line that we have used extensively in the past and have shown are a good model for placenta. We will overexpress HFE using transfection and will use siRNA to knock-down the peptide. We will then measure 59Fe uptake using 59Fe-125Tf. The effect of hepcidin can be tested using synthetic peptide, with the significance of different amino acids being tested, if appropriate by substitution with ones of similar or different properties. We will carry out dose response experiments in cells that have been transfected with HFE or which have had endogenous levels reduced by siRNA. These experiments will determine whether there is a direct interaction between the proteins and how they are related to each other. The data will clarify significantly the role that liver HFE and hepcidin play in the regulation of Fe transfer, at least during pregnancy, and will provide valuable insights into the mechanisms underlying their role.

Summary

The WHO states that, during pregnancy, 20 per cent of the women in UK have anaemia greater than the clinically normal anaemia of pregnancy. This has important consequences for both the mother and her foetus. The placenta responds by up-regulating proteins of Fe transfer to try and minimise the problem, but how it senses Fe status is unknown. Recently two new sensors of body iron homeostasis, HFE and hepcidin, have been identified. Mutations in these genes cause Fe overload, but how they work and how, or whether, they interact is still unknown. We will study how the maternal, foetal and placental HFE and hepcidin interact, how they regulate Fe transfer in the placenta and whether hepcidin in the mother could be used as a biomarker of her Fe status rather than the poor ones used at present. To do this, we will use mice in which the HFE gene has been knocked out. We will measure Fe transfer and the responses of the genes of iron transfer (TfR, DMT1, IREG1 and eleutherin) in relation to the mother and to the genotype of her foetuses. If maternal HFE is critical, we would expect a diminution of the responses in all offspring, irrespective of their own genotype. If the foetal genotype is most important, we would expect graded responses depending on the genotype. We will examine the role of hepcidin by injecting into Fe deficient mice, in which endogenous peptide will be reduced. Although this will not differentiate between direct and indirect effects, it will show whether the peptide plays an integral role. A closer investigation of the interaction between the two proteins will be carried out using the placental cell line BeWo, a good model for Fe transfer. We wil modulate HFE expression by transfection and by siRNA, Fe uptake using 59Fe-transferrin and gene expression using real-time PCR.

Committee	Closed Committee - Animal Sciences (AS)
Research Topics	Diet and Health
Research Priority	X – Research Priority information not available
Research Initiative	X - not in an Initiative
Funding Scheme	X – not Funded via a specific Funding Scheme

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