Previously, we identified the heavy chain of ferritin as a developmentally regulated nuclear protein of embryonic chicken corneal epithelial cells. The nuclear ferritin is assembled into a supramolecular form indistinguishable from the cytoplasmic form of ferritin found in other cell types and thus most likely has iron-sequestering capabilities. Free iron, via the Fenton reaction, is known to exacerbate UV-induced and other oxidative damage to cellular components, including DNA. Since corneal epithelial cells are constantly exposed to UV light, we hypothesized that the nuclear ferritin might protect the DNA of these cells from free radical damage. To test this possibility, primary cultures of cells from corneal epithelium and stroma, and from skin epithelium and stroma, were UV irradiated, and DNA strand breaks were detected by an in situ 3'-end labeling method. Corneal epithelial cells without nuclear ferritin were also examined. We observed that the corneal epithelial cells with nuclear ferritin had significantly less DNA breakage than other cell types examined. Furthermore, increasing the iron concentration of the culture medium exacerbated the generation of UV-induced DNA strand breaks in corneal and skin fibroblasts, but not in the corneal epithelial cells. Most convincingly, corneal epithelial cells in which the expression of nuclear ferritin was inhibited became much more susceptible to UV-induced DNA damage. Therefore, it seems that corneal epithelial cells have evolved a novel, nuclear ferritin-based mechanism for protecting their DNA against UV damage.

Previously, we generated monoclonal antibodies against chicken corneal cells (Zak, N. B., and Linsenmayer, T. F. (1983) Dev. Biol. 99, 373). We have now observed that one group of these antibodies reacts with a developmentally regulated component of corneal epithelial cell nuclei. This component is the heavy chain of ferritin, as determined by analyses of immunoisolated cDNA clones and immunoblotting of the protein. Immunoblotting also suggests that the nuclear ferritin may be in a supramolecular form that is similar to the ironbinding ferritin complex found in the cytoplasm of many cells. In vitro cultures and transfection studies show that the nuclear localization depends predominantly on cell type but can be altered by the in vitro environment. The appearance of nuclear ferritin is at least partially under translational regulation, as is known to be true for the cytoplasmic form of the molecule. The tissue and developmental distributions of the mRNA for the molecule are much more extensive than the protein itself, and the removal of iron from cultures of corneal epithelial cells with the iron chelator deferoxamine prevents the appearance of nuclear ferritin. At present the functional role(s) of nuclear ferritin remain unknown, but previous studies on cytoplasmic ferritin raise the possibility that it prevents damage due to free radical generation ("oxidative stress") by sequestering iron. Although it remains to be tested whether nuclear ferritin prevents oxidative damage, we find this an attractive possibility. Since the corneal epithelium is transparent and is constantly exposed to free radical-generating UV light, it is possible that the cells of this tissue have evolved a specialized mechanism to prevent oxidative damage to their nuclear components.Nuclei contain a myriad of different proteins as well as nucleic acids. Nuclear proteins range from general ones, such as the histones that are universally present in cells and determine the structure of chromatin, to the DNA-binding regulatory proteins, some of which are cell-specific and developmentally regulated. In the present investigation, we provide evidence that within the corneal epithelium of embryonic chicks, ferritin is also a developmentally regulated nuclear protein. In other cell types that have been investigated, the molecule is cytoplasmic and is thought to function as a protective antioxidant by sequestering iron (1, 2) (see "Discussion").In earlier work we sought to identify developmentally regulated components of the embryonic avian cornea by generating hybridomas against corneal cell suspensions (3). By immunofluorescence, one group of antibodies (including antibody 6D11) reacted with a corneal epithelial antigen but had little or no detectable reactivity with other tissues in the embryo. During development, this antigen first became detectable by immunofluorescence in 12-day corneas, the time when the epithelium begins to stratify (4). We also found it to arise de novo in cultured corneas, and it has been used as a marker for corneal epithe...

Previously we reported that ferritin in corneal epithelial (CE) cells is a nuclear protein that protects DNA from UV damage. Since ferritin is normally cytoplasmic, in CE cells, a mechanism must exist that effects its nuclear localization. We have now determined that this involves a nuclear transport molecule we have termed ferritoid. Ferritoid is specific for CE cells and is developmentally regulated. Structurally, ferritoid contains multiple domains, including a functional SV40-type nuclear localization signal and a ferritin-like region of ϳ50% similarity to ferritin itself. This latter domain is likely responsible for the interaction between ferritoid and ferritin detected by co-immunoprecipitation analysis. To test functionally whether ferritoid is capable of transporting ferritin into the nucleus, we performed cotransfections of COS-1 cells with constructs for ferritoid and ferritin. Consistent with the proposed nuclear transport function for ferritoid, co-transfections with full-length constructs for ferritoid and ferritin resulted in a preferential nuclear localization of both molecules; this was not observed when the nuclear localization signal of ferritoid was deleted. Moreover, since ferritoid is structurally similar to ferritin, it may be an example of a nuclear transporter that evolved from the molecule it transports (ferritin).

We previously obtained evidence that ferritin is a nuclear protein in embryonic avian corneal epithelial (CE) cells, and that the ferritin in this site protects DNA from UV-induced damage. UV irradiation is known to produce reactive oxygen species (ROS) and ferritin is known to ameliorate further oxidative damage by sequestering free iron, thus decreasing the formation of hydroxyl radicals through the Fenton reaction. Here we present evidence that nuclear ferritin can similarly prevent damage by the ROS, H 2 O 2 . These results, when coupled with our previous ones showing that nuclear ferritin appears in the CE early in its development, raises the possibility that this ferritin may serve two protective roles. The initial one would be during embryonic development to protect the CE from ROS endogenously produced by the embryo itself (e.g., H 2 O 2 ; the latter one would be post-hatching to protect the CE from environmentally produced oxidative insults (e.g., from U.V. light).

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