Molecular and cellular insights into iron regulation

The iron hormone hepcidin is regarded as the main iron homeostatic regulator in the human body. It is predominantly produced by hepatocytes in response to systemic iron excess. However, since the cellular and molecular mechanisms involved in hepcidin expression are not fully understood, this project involved studying hepcidin expression and the role of the pro-region of the hepcidin prohormone in regulation of iron homeostasis. Iron overdose in Chinese hamster ovary-transferrin receptor variant (CHO TRVb1) cells resulted in increased hepcidin peptide secretion after 30 min and 2 hours (p<0.03) as well as 24 and 48 hours (p<0.01). Also, partial characterisation of the previously unknown CHO-gene sequences of Hfe, Slc40-a1 and Irp2, was achieved. To determine the effect of intracellular iron overload on hepcidin expression, recombinant transferrin receptor 1 (rec-TfR1) HepG2 cells were created which express modified TfR1 to maximise iron uptake. Upon holotransferrin (5 g/L) treatment these cells showed significantly increased iron uptake which was in contrast to the response by Wt HepG2 cells. Also, it was shown for the first time that hepcidin peptide secretion increased upon iron overdose to HepG2 cells after 30 min, 2,4,24 and 48 hours (p<0.05). Also, holotransferrin treatment (5 g/L) increased hepcidin mRNA levels; in Wt HepG2 cells by 0.6 fold (on average) after 30 min, 2,4,6 and 24 hours and in rec-TfR1 HepG2 cells by 0.5 fold after 2 h (p<0.02). Gene expression studies of TfR1, SLC40-A1, and HFE upon iron overdose showed opposing functionalities of TfR1 and SLC40-A1 in maintaining intracellular iron homeostasis and emphasised the significance of HFE in hepcidin induction. Additionally, localisation studies with the pre-pro derivative of preprohepcidin identified its presence in the nucleus, suggesting its involvement in the gene regulation process and thus possible participation in maintaining iron homeostasis. In conclusion, rec-TfR1 HepG2 cells partially resemble haemochromatotic cells and the findings indicate that hepcidin regulation involves the interaction between several iron-related genes and the extracellular and intracellular iron levels.