Ceruloplasmin Alters Intracellular Iron Metabolism and Increases Ferritin Levels and Glutamate Secretion in Lens Epithelial and Retinal Pigmented Epithelial Cells

Abstract

Purpose: : Ceruloplasmin (Cp) is a ferroxidase responsible for facilitating the exodus of iron from cells. In humans, aceruloplasminemia results in iron accumulation in the brain and retina associated with neural and retinal degeneration. We have previously shown that Cp increases iron efflux from and iron incorporation into ferritin in lens epithelial cells (LEC). The present study is focused on a determination of the effects of Cp on intracellular iron metabolism in LEC and retinal pigmented epithelial cells (RPE).

Methods: : RPE and LEC were obtained from eyes of dogs euthanized at a local pound. Cells were cultured until confluent and Cp in serum-free cell conditioned medium (CCM) was determined by Western blot. Ferritin in cell lysates was determined by ELISA. Cells were incubated with ⁵⁹Fe-transferrin and ⁵⁹Fe was measured in membrane pellets and cytosol. Glutamate in CCM was measured using an Amplex Red kit (Invitrogen).

Results: : Both LEC and RPE synthesize and secrete Cp. Addition of exogenous Cp increased ferritin synthesis (79%) and the concentration of ferritin (35%) in LEC, indicating an increase in available iron in these cells. When LEC were loaded with ⁵⁹Fe-transferrin, Cp caused an increase in the amount of ⁵⁹Fe in the membrane pellet (34%). In a previous study, increased iron availability in LEC and RPE caused an increase in glutamate synthesis and secretion by increasing the activity of cytosolic aconitase. In the present study, Cp increased glutamate secretion by RPE (32%) and LEC (50%) and this effect was blocked by the aconitase inhibitor oxalomalate. Immunofluorescent labeling of Cp in RPE and LEC indicated that Cp is normally present throughout the cytoplasm, but was also found in the nucleus in a punctuate pattern.

Conclusions: : Our data indicate that exogenously added Cp has profound effects on iron movement between intracellular compartments. These include an increase in the labile iron pool, which increased ferritin synthesis in LEC, and glutamate secretion by LEC and RPE. The increase in labeled iron movement from the cytoplasm into the membrane fraction confirms our earlier findings of increased iron exodus from lens cells by exogenously added Cp. Cp has important roles in regulating iron metabolism within the cell and its absence results in retinal pathology as demonstrated in Cp/hephaestin knockout mice.

Keywords: metabolism • oxidation/oxidative or free radical damage • retinal pigment epithelium