Abstract
Ultraviolet (UV) light constitutes a major environmental hazard for all exposed tissues of the body. UV light can damage a variety of macromolecules—including alterations to DNA that are potentially carcinogenic. Much of the UV-induced damage to DNA is thought to result from the generation of reactive oxygen species (ROS) and the subsequent conversion of lowly-energetic ROS (e.g., H2O2) to highly energetic hydroxyl radicals. This occurs through the iron-mediated Fenton reaction. In skin, UV-induced damage to DNA is thought to be a major factor in the increasing incidence of epidermal cancers. However, corneal epithelial (CE) cells seem to be refractory to such damage—as primary cancers of these cells are extraordinarily rare—even though this tissue is transparent and constantly exposed to UV light. This suggests that CE cells have evolved a defense mechanism(s) that prevents damage to their DNA produced by both UV irradiation and ROS (e.g. H2O2). Studies in our laboratory suggest that one such mechanism involves having the iron-sequestering molecule ferritin in a nuclear localization—rather than cytoplasmic location it typically has in other cell types.
Other studies show that the subunit of this nuclear ferritin is a “typical” H-chain that is transported into the nucleus by a CE-specific nuclear transporter—we have termed ferritoid for its similarities to a ferritin subunit. Ferritoid has two functional domains. One is similar to ferritin and most likely facilitates binding to ferritin subunit(s); the other has a consensus SV40-like nuclear localization signal that is responsible for nuclear transport. Other studies show that ferritoid not only effects nuclear transport, as within the nucleus it remains associated with ferritin—participating in the formation of unique heteropolymeric complex(es). These complexes have unique structural and functional properties. These include a size which is half that of a “typical” cytoplasmic ferritin, a low content of iron, and the ability to bind to DNA—all of which may contribute to the prevention of damage to DNA.
Also, from studies on developing corneas, we have determined a number of additional properties of ferritoid and its interaction with ferritin. These include that: (1) temporally the synthesis of ferritoid and ferritin proteins are closely regulated—several hours before ferritin, (2) that iron and thyroxine are involved in regulating the synthesis of both ferritin and ferritoid, (3) the nuclear transport activity of ferritoid requires an interaction with ferritin, and (4) the interaction between ferritoid and ferritin involves phosphorylation of ferritoid.
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Notes
- 1.
Several other studies have reported ferritin in a nuclear location in other cell types, including nucleated red blood cells and cells in developing rat brain, as well as astrocytoma and glial cell lines, and cells subjected to iron overloading and other pathological conditions.
- 2.
The ISEL method [8, 14] is based on UV-induced ROS damage to DNA activating an excision-repair system [20, 28]. This temporarily produces DNA breaks that can be detected by 3′-end labeling. Also, when necessary the fluorescent signals can be quantified—as the percentage of cells showing a positive signal, and the strength of the signal produced [11].
- 3.
At the time these studies were done, there was no antibody against ferritoid—thus the need to use epitope tags.
- 4.
As this uniform distribution is also found in single transfections for ferritin, we think that this reflects the over-expression of ferritin by the transfected construct is more rapid than the assembly of the supramolecular ferritin complexes. Therefore some of the newly-synthesized monomers, and low molecular weight complexes are able to diffuse into the nucleus.
- 5.
By the time these analyses were undertaken, we had an anti-ferritoid antibody [6].
- 6.
In these studies, another potentially interesting observation concerns the cellular relationship between ferritoid and cytokeratin K3 (a marker for CE cell differentiation). In the central cornea—where all the CE cells are mature—ferritoid and K3 are both found in all cells. However, at the periphery, where the CE cells are undergoing their initial differentiation, ferritoid is present in basal CE cells that are not yet synthesizing detectible K3. Thus, the synthesis of ferritoid may represent an early event in the differentiation of CE cells. In addition, this observation reinforces the potential importance of ferritoid in the protection of the DNA of cells from damage—especially if the cells that have ferritoid, but no K3, are CE stem cells.
- 7.
The CAM is comprised of all the extraembryonic membranes of the developing embryo. It supports the full-range development of an explanted cornea—even if the explants are from early stage embryos [50].
- 8.
As primary cultures of pre-ferritin stage CE cells sometimes do not provide sufficient material for certain types of analyses, whole corneal organ cultures were employed (i.e., corneas floating in medium). The behavior of the CE of such corneas was identical to that of CE cells in primary culture, and the quantities of CE harvested from such cultured corneas [using treatment with the enzyme Dispase [45] easily provides sufficient CE tissue for assays most assays (e.g., qRT-PCR, protein determinations, and microarrays.
- 9.
However, these in vitro studies also showed that ferritoid undergoes upregulation at the translational level—as the stimulation of mRNA synthesis, in itself, does not account quantitatively for the magnitude of the increase in ferritoid protein. Thus, for ferritoid it seems that following synthesis of its RNA, translational upregulation regulation also becomes important. (For a more detailed discussion of this see [6]).
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Acknowledgement
This work was supported by NIH Grant R01EY013127 to TFL.
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Linsenmayer, T.F. et al. (2015). Corneal Epithelial Nuclear Ferritin and Its Transporter Ferritoid Afford Unique Protection to DNA from UV Light and Reactive Oxygen Species. In: Babizhayev, M., Li, DC., Kasus-Jacobi, A., Žorić, L., Alió, J. (eds) Studies on the Cornea and Lens. Oxidative Stress in Applied Basic Research and Clinical Practice. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-1935-2_3
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