Is epiplakin a true keratin chaperone?

The protein epiplakin is characterized by its unusual size and structure. It is only found in epithelial cells where it binds to keratins, which are proteins forming cytoskeletal filaments. Mice in which epiplakin was removed from the body by the use of genetic techniques did not show any obvious differences compared to normal mice. However, when these mice were compared with normal mice in regard to their susceptibility for different stress conditions in pancreas and liver, they clearly show increased tissue damages in those diseases which lead to a significantly upregulation of epiplakin and keratin expression. In addition, pancreata and livers devoid of epiplakin showed an increased number of cells displaying keratin aggregates, indicating impaired disease-induced keratin network reorganization. In accordance with these data, cells isolated from epiplakin-free livers showed an increased number of keratin aggregates in combination with cell death when keratin overexpression was induced. This effect could be inhibited by administration of a substance which prevents the formation of keratin aggregates. These studies demonstrate that during different forms of experimental injuries of the pancreas and liver, epiplakin plays a protective role by controlling disease- induced keratin reorganization. We have strong evidence that the enhanced formation of keratin aggregations in the absence of epiplakin is the reason for the aggravated course of stress-induced organ injuries in mice lacking epiplakin. These data suggest that epiplakin is a tailored guidance protein for keratins during their reorganization in simple epithelia. In this project, we want to further elucidate these proposed functions of epiplakin in simple epithelia by performing a combination of approaches using genetically altered mouse lines, microscopical analyses of cultured cells, and biochemical methods. We plan to generate mouse lines devoid of epiplakin which express different levels of keratin to investigate whether keratin expression levels are affecting the problems observed in epiplakin-deficient mice. For the microscopy part, the main goal is to visualize full length epiplakin and keratins in living cells to monitor their localization and movement. In addition, we will perform interaction studies with epiplakin and keratins in test tubes. I expect to be able to reveal how epiplakin fulfils its proposed keratin-guiding functions. Such data would significantly increase our knowledge about the mechanisms responsible for the formation of keratin filaments and their breakdown, which are to date not as well understood as those for other filament types existing in cells. Though, until now no human patients could be found showing mutations in their epiplakin genes, I expect that our findings will trigger the search for such individuals. Our data could finally prove that the absence of epiplakin might hold responsible for the adverse outcome of various pathologies of the gastrointestinal tract.

Keratins are forming filaments in the inner part of cells of most animals. They exert important functions like strengthening the cells when they are exposed to various forms of stress. Keratins are not simply rigid "cables", they also have to be able to disassemble when cells must adapt to special conditions. The protein epiplakin is characterized by its unusual size and structure. It is only found in epithelial cells where it colocalizes with keratin filaments. Biochemical experiments showed that epiplakin binds to keratins. Genetically modified mice, devoid of epiplakin showed any obvious differences compared to normal mice. However, when these mice were compared with normal mice in regard to their susceptibility for different stress conditions in pancreas and liver, they clearly show increased tissue damages in certain diseases. In addition, pancreata and livers devoid of epiplakin showed an increased number of cells displaying keratin aggregates, indicating impaired disease-induced keratin network reorganization. These studies demonstrate that during different forms of experimental injuries of the pancreas and liver, epiplakin plays a protective role by controlling disease-induced keratin reorganization, but the mechanism behind is still unclear. In this project, we could show that epiplakin can be found in all animals having a spine like fishes, frogs, lizards, birds and mammals. This finding supports our assumption that epiplakin has important functions for all these different species. In addition, we gained experimental data that will help to reveal the conditions in which epiplakin functions are beneficial for organisms. Using live cell imaging of cells expressing epiplakin and keratin both tagged with fluorescent proteins, we identified an unexpected and yet undemonstrated diffuse intracellular localization of epiplakin under normal conditions. This state quickly changed upon induction of several kinds of stress like irradiation with ultraviolet light, which induced a perfect colocalization of epiplakin with keratin filaments. The re-localization of epiplakin was reversible and dependent on elevation of Ca2+ levels in the cell. This characteristic of epiplakin was previously overlooked due to its complete keratin association in microscopic images, when cells are prepared for standard microscopy and thus killed. Moreover, we could show that keratin filament association of epiplakin led to a significantly reduction of keratin filament dynamics. Thus, we propose that epiplakin stabilizes the keratin network in stress conditions, which involve increased cytoplasmic Ca2+.