Abstract
Purpose::
As the cornea is constantly exposed to environmental insults, such as ultraviolet (UV) light, it has evolved mechanisms to protect itself. Our previous studies identified a novel protective mechanism in the corneal epithelium (CE) involving the presence of ferritin in the nucleus, where our studies suggest it protects DNA from UV-induced damage. Although ferritin does not have a nuclear localization sequence, it is efficiently transported into the nucleus in CE cells. We have observed that this nuclear transport is mediated by a novel, tissue-specific protein, ferritoid, that binds to ferritin and carries it into the nucleus. In the present study we have examined the mechanisms involved in regulating the ferritoid-mediated nuclear transport of ferritin.
Methods::
To study the expression of ferritoid and ferritin during development, RT-PCR and immunofluorescence analyses were performed at a range of stages during corneal development. To examine the appearance of these molecules in primary cultures of CE cells, the iron chelator deferoxamine was used to inhibit the expression of ferritoid and ferritin. Then cells were allowed to recover for various lengths of time in high iron medium, and the expression of ferritoid and ferritin was analyzed by immunofluorescence. To investigate the role of phosphorylation on transport, phosphorylation of ferritoid in CE cells was first examined by cell fractionation, immunoblotting, and mass spectrometry. Then cell cultures were transfected with ferritoid constructs containing mutated phosphorylation sites and analyzed for the localization of ferritoid and ferritin by immunofluorescence.
Results::
Both in CE tissue and in CE cell cultures treated with deferoxamine and allowed to recover over time, ferritoid appears before ferritin and then remains in the cytoplasm until ferritin is expressed, suggesting that ferritoid requires ferritin binding to initiate nuclear transport. Other studies suggest that differential phosphorylation of ferritoid may be involved in its binding to and subsequent transport of ferritin. Immunoblot analyses of fractioned CE cell lysates show that ferritoid when bound to ferritin is phosphorylated but that the ferritoid monomer is not. Consistent with this, mutation of certain ferritoid phosphorylation residues inhibits ferritin transport in cultured cells.
Conclusions::
Nuclear transport of ferritin by ferritoid requires the expression of both proteins and is modulated by differential phosphorylation of ferritoid.
Keywords: cornea: epithelium • development • phosphorylation