Effect of copper chelation on hepcidin expression in the human hepatoma cell line, HepG2

Hepcidin, a peptide hormone produced mainly by the liver,1 has an essential role in iron homeostasis. It is produced as prepropeptide of 84 amino acids and, after two consecutive cleavage, a form of 25 amino acids is secreted in the bloodstreem.2-4 Hepcidin is able to control the body iron concentration by the binding to the only known protein involed in iron export from the cells, ferroportin, that is present on the basolateral membrane of enterocytes, on macrophages, hepatocytes and other cells types.5,6 After the binding, ferroportin is degraded and the iron uptake by the diet and iron recycling from senescent red blood cells, is inhibited.7 View the important role of hepcidin in iron metabolism, its expression is strictly regulated; in fact, dysfunctions or mutations that modify its expression, lead to different pathologies, such as hemochromatosis and anemia.8-10 Some crystallographic studies have demonstrated that an other important metal for the life of all the organisms, copper, could be important for hepcidin funtion; in fact, the presence of copper in solution can influence the aggregation state of hepcidin and studies in which a cell line was used as model system, have highlighted the importance of this element in the capacity of hepcidin to induce ferroprtin degradation.11,12 The link between copper and iron is demonstarated also by an other important plasma protein, ceruloplasmin, a ferroxidase enzyme that contain copper in the catalytic site and is responsable of the transport of 90% of plasma copper13 Different studies have demonstrated that animals fed with a copper deficient diet, have low levels of plasma ceruloplasmin and, its characteristic oxidase activity, is strongly reduced;14,15 this kind of situation is normally present in Wilson disease patients, in which a defect in copper transport in the Golgi, is cause of the lack of plasma ceruloplasmin.16 To better understand if the copper has a role on hepcidin expression, we have used a human hepatoma cell line, HepG2, to investigate the influence of copper chelation and inflammation, by the use of the citokine interleukin-6,17 on the level of hepcidin expression. To corroborate our results, we have also studied ceruloplasmin expression. Our results have demonstarted that the use of the chelation substrate, Bathocuproinedisulfonic acid (BCS) has a slight effect on ceruloplasmin mRNA levels, both in normal and stimulated IL-6 cells, while the intracellular level of protein is markedly reduced when BCS is added in both conditions. The inhibitory effects is more evident if the secreted form of the protein is considered; in fact, western blot and in gel oxidase activity analysis, have shown a disappearence of the protein and of its activity in the growth cellular media, results coherent with bibliographic data. The effect on hepcidin is also evident. Even if no statistical significative differences were found at mRNA level, the effect on the concentration of the intracellular prepropeptide is more evident, especially when the citokine and BCS are used togheter. The presence of both substrates leads to a decrease of the protein level when it is compared to the use of interleukin-6 alone and this effect was more evident when the concentration of the peptide in the cellular growth media was assayed. Our results demonstrate that a copper deficiency could negatively influence hepcidin expression even if other studies are necessary to better understand this mechanism.