Effects of UV Light on Ferritin Synthesis, Ferritin Assembly and Fe Metabolism in Lens Epithelial Cells

Abstract

Abstract: : Purpose: The multimeric Fe storage protein, ferritin, can protect cells against oxidative stress by safely storing potentially reactive Fe. Its synthesis is regulated by Fe and redox conditions. We have previously shown that UVB irradiation significantly increased ferritin synthesis. It was the purpose of this investigation to determine how increased ferritin synthesis affected Fe metabolism in cultured lens epithelial cells (LEC). Methods: Primary cultures of canine LEC were exposed to UVB irradiation (20mJ/cm₂ ) in the presence or absence of ascorbic acid (0.15 mM). De novo ferritin synthesis was measured by ₃₅S-methionine labeling, immunoprecipitation and gel electrophoresis. Fe uptake into ferritin was determined by incubating the cells with ₅₉Fe-transferrin, then acetone precipitation of cytosolic proteins and native PAGE electrophoresis. Assembly of holoferritin was determined by ₃₅S-methionine labeling and native PAGE electrophoresis. Radioactivity in the gels was determined electronically (Instant Imager, Packard Canberra). Results: UVB irradiation increased synthesis of ferritin two-fold and was additive to the two-fold increase in ferritin synthesis induced by ascorbic acid. However, while ascorbic acid significantly increased Fe incorporation into ferritin (43%, p<0.5), UVB did not alter Fe incorporation into this protein. In addition, total ferritin concentration in the cytosol was increased by ascorbic acid, but not UV irradiation, despite the increase in de novo ferritin synthesis caused by this treatment. Using native gel electrophoresis we have determined that, while UV light increases synthesis of ferritin subunits, they are not assembled into holoferritin molecule. In contrast, the ascorbic acid induced increase in subunit production was reflected in an increase in newly assembled protein. Conclusion: Ferritin is made up of 24 subunits of two different sizes and functionality. We have demonstrated that UV light can induce the synthesis of both subunits, but that they do not assemble into a functional ferritin molecule under these conditions. There is little known about the process of assembly of ferritin, perhaps the effects of UV light on this process can be used to help dissect its underpinnings.

Keywords: 504 oxidation/oxidative or free radical damage • 537 radiation damage: light/UV • 527 protein structure/function