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Mammalian regulation of iron circulation

Final Activity Report Summary - MARIC (Mammalian Regulation Of Iron Circulation)

In my laboratory we studied the mechanisms and regulation of systemic iron circulation. Iron is an essential nutrient for mammalians, but becomes toxic when at excess and not properly handled or stored. It is efficiently recycled and systemic and cellular equilibrium is achieved by tight regulation of iron uptake, release, storage and transport.

The European Research Council (ERC) starting grant focussed on two projects within this subject. Firstly, the immunologic effects of red blood cell recycling were examined. The major active pool of iron in the body is present in red blood cell (RBC)-hemoglobin. When RBCs age they are removed by macrophages, a cell type that is part of our immune system and is in charge for the removal, i.e. phagocytosis, and degradation of any particle, pathogenic or not, that could be recognised as ‘unwanted’. Usually, following a phagocytosis event, macrophages undergo an activation program that is able to efficiently kill an ingested pathogen, but very little is known on macrophage reactions following RBC ingestion.

One macrophage reaction to RBC ingestion is the up-regulation of the enzyme heme-oxygenase, which is responsible for the degradation of heme, the oxygen carrying molecule in hemoglobin. We determined, using a fluorescent protein protection assay, the exact location of the active site of heme-oxygenase and we could show that, during RBC-phagocytosis, heme-oxygenase was brought into close vicinity of the ingested RBC.

Interestingly, a shorter form of the enzyme was expressed in response to RBC ingestion; therefore we also studied the characteristics and possible functions of this short form of heme-oxygenase. In addition, we developed a system to age RBCs in vivo and we could thus mimic RBC-phagocytosis by macrophages in a test-tube. This enabled us to study immunologic effects of RBC-phagocytosis on macrophages on a global level, and we were analysing these effects by the time of the project completion.

Secondly, the mechanisms of trafficking of the iron storage protein ferritin were investigated. Ferritin is a large protein, built of 24 subunits that spontaneously assemble to a hollow sphere, capable of storing many atoms of iron in a soluble and non-toxic way. In mammals, ferritin is usually an intracellular protein, however little is found in the plasma and the way in which ferritin reaches the blood stream is unknown.

We studied the characteristics of plasma ferritin so as to get clues on how ferritin may be secreted and elucidate whether ferritin could be a molecule that was involved in iron transport. We found that plasma ferritin contained little of the subunit needed for iron loading and, furthermore, contained little iron. Characteristics of classically secreted proteins, i.e. N-glycosylation patterns, were under investigation by the time of the project completion to give us a lead to look for mechanisms of its secretion.

In addition, ferritin secreted from macrophages also contained iron and the subunit needed for iron loading and was insensitive to an inhibitor of the classical secretion pathway. Taken together, these results suggested that ferritin might under certain conditions play a role in local iron distribution within a tissue, but, most likely, not in systemic iron distribution. The understanding of mechanisms and regulation of ferritin transport and secretion were important for the understanding of the role of this secreted form of ferritin.