Ceruloplasmin is Made and Secreted by Lens Epithelial Cells and Alters Iron Availability

Abstract

Abstract: : Purpose: Ceruloplasmin (Cp), generally considered to be an extracellular protein has ferroxidase activity which converts ferrous to ferric iron, the form which binds to transferrin (Tf). When bound to Tf, iron does not cause oxidative damage. There is evidence that Cp can influence iron uptake by cells and its storage in ferritin. Thus, it is not surprising that Cp and Tf have antioxidant activity. It was the purpose of this study to determine if lens epithelial cells (LEC) make and secrete Cp and if Cp and Tf affect iron availability. We have previously shown that Tf is made and secreted by the lens. Methods: Cultured canine LEC were used in this study. LEC were cultured in MEM + 10% serum. At the time of the experiments the culture medium was changed to MEM without serum. LEC were loaded with ⁵⁹Fe–Tf for 24h and then treated with Cp, apoTf (contains two free iron binding sites) or Cp and apoTf for an additional 48h. At this time, the amount of Fe found in ferritin and a low molecular weight pool (LMWP) in the cells and the media was determined. In another set of experiments, Cp, apoTf or Cp and apoTf treated cells were loaded with the fluorescent iron chelator calcein and the amount of chelatable (not bound to protein) iron was measured. Western analysis was used to determine the amount of Cp in the LEC and in the media after 24 h incubation. Results:Cp significantly increased Fe incorporation into intracellular ferritin, but had no effect on the size of a LMWP of Fe in LEC. Cp also had no effect on extracellular Fe distribution. However, when apoTf was added after Fe loading, the extracellular LMWP of iron was reduced to near zero and Fe in the Tf band increased dramatically, indicating that Fe in the LMWP was chelatable by Tf and that apoTf could retrieve Fe from the cell. In the case of intracellular chelatable iron measured by calcein, Cp caused a 10% increase, apoTf a 37% increase and Cp + apoTf a 49% increase. Western blots of LEC cytosol and media from cells incubated in serum free media for 24h indicates that Cp is present intracellularly and is secreted into the media in large quantities. Conclusions: Cp and Tf are made and secreted by LEC and have profound effects on intracellular iron movement. This intracellular effect was surprising since Cp and Tf are considered to be extracellular proteins. This has important implications for many tissues as the regulation of iron movement between intracellular compartments is poorly understood. Additionally, aceruloplasminemia can cause numerous pathologies related to disturbances in iron metabolism. Clearly, a better understanding of Cp’s role in intracellular iron metabolism is needed.

Keywords: metabolism • antioxidants • oxidation/oxidative or free radical damage