Abstract
Purpose:
The efflux of iron from cells is facilitated by ferroportin (FPN), the only documented mammalian iron exporter protein. The presence of excess iron in RPE found in ocular pathologies such as AMD may result from the inability of cells to efficiently remove iron via FPN. We have recently shown that knockdown of FPN in primary canine RPE cells resulted in decreased Fe efflux, but we had not characterized changes in FPN expression and localization caused by treatments mimicking pathologically-relevant conditions. FPN is expected to be found in the cell membrane, but we also investigated whether FPN localizes to organelle membranes, and whether the distribution of FPN changes under conditions of altered iron status and hypoxia. To better characterize the normal sorting pathway and targeting of FPN in a model that better reflects the in vivo condition, we also examined FPN localization using a tight-junctional, polarized RPE cell culture system.
Methods:
Polarized and non-polarized primary canine RPE were treated with 10µg/ml ferric ammonium citrate (FAC), 30µM Dp44mT chelator, or hypoxic conditions (0.5% O2) for 24 or 48 hours and then either processed for Western analysis of FPN, or fixed and fluorescently immunolabeled for FPN. CellLight® organelle markers were used for colocalization studies.
Results:
Western analysis showed that treatment of RPE with iron resulted in a 50% increase in FPN expression, while depletion of iron resulted in a 50% decrease in FPN levels. Interestingly, hypoxia also caused a decrease in FPN expression. Changes in overall cellular immunofluorescence reflected Western results, but we were also able to visualize FPN in the golgi and early and late endosomes. In polarized RPE cells, FPN was localized to both apical and basolateral surfaces.
Conclusions:
Changes in the iron and oxygen status of RPE cells affect FPN expression and localization. FPN was localized to the plasma membrane, where it would be expected to function in removal of excess iron from the cell, but also in organelles, suggesting that FPN may also function in the redistribution of iron between different membrane-bound compartments of the cell, thereby impacting overall cellular iron status. These studies will be useful to further characterize the dynamics of iron metabolism in RPE cells under normal and pathological conditions.