Epiplakin in simple epithelia

Epiplakin is a member of a family of cytoskeletal linker proteins called plakins. Compared to the other protein family members it has an unusual structure comprising of solely 16 (mouse) or 13 (human) plakin repeat domains. Epiplakin expression is restricted to epithelial tissues and it binds to intermediate filaments, mainly to keratins, which are the only binding partners identified so far. Epiplakin-deficient mice did not show any discernible phenotype, except for a slight acceleration of keratinocyte migration. These findings are in clear contrast to other proteins belonging to the plakin protein family like plectin, desmoplakin, and BPAG1, which play an important role in mechanically strengthening the skin as shown by phenotypes of knock-out mice. Subsequent experiments using primary keratinocytes from epiplakin-deficient mice showed that the biological role of epiplakin seems to be different from these plakins and to be rather connected with cellular stress response. This protective function appears to be more prominent in simple epithelia tissues as shown by the knock-down of epiplakin in HeLa cells, which in contrast to that in keratinocytes caused disruption of intermediate filament networks. However, a comprehensive analysis of the in vivo function of epiplakin in simple epithelia using defined animal models is still missing to date. In preliminary pancreatitis induction experiments we could show that epiplakin knock-out animals suffered from a more severe course of disease compared to control animals. In order to further elucidate the biological function of epiplakin in simple epithelia, I propose to perform a combination of experiments using mouse injury models and experiments based on cell culture, biochemistry and video microscopy. In the mouse I plan to use several stress models for simple epithelia in different organ systems which will be complemented by experiments with primary cells. As the use of a pan-creatitis model in epiplakin deficient mice already resulted in a phenotype, I will start with a more de-tailed analysis of epiplakin function in pancreas. Two additional organs, liver and intestine, were chosen first based on the availability of established injury models and second motivated by the fact that mutations of the epiplakin binding protein keratin 8 in mice resulted in clear phenotypes in these organs. One additional reason to choose the liver model is the possibility to isolate primary hepatocytes for a further molecular analysis of epiplakin function. These studies will be complemented by biochemical and cell culture based methods to investigate epiplakin interaction with simple epithelial keratins in more detail and to reveal epiplakin functions in keratin network recovery after stress. I expect to be able to show that epiplakin has a vital role as a stress response protein in simple epithelia, for example by reorganizing keratin filament networks after stress induced collapse or depolymerization. Besides providing further insight in the biological function of epiplakin this work might open entirely new perspectives in the understanding of particular stress responses in various organs harboring simple epithelia. Apart from increasing our knowledge about stress response in these tissues, the relevance of the proposed study is its implication for molecular deficits causing common human disorders like chronic liver diseases, chronic pancreatitis, and inflammatory bowel disease.

The protein Epiplakin is characterized by its unusual size and structure. The only binding partners of epiplakin identified so far are keratins, proteins which form cytoskeletal filaments in epithelia. To reveal the yet unknown function of epiplakin in simple epithelia, its expression pattern in pancreas, liver and intestine was analyzed, both at normal conditions and during stress. In addition, healthy mice and mice in which epiplakin was removed from the body by the use of genetic techniques were compared in regard to their susceptibility for different stress conditions in these organs. Epiplakin was shown to be expressed in duct cells and acinar cells of the pancreas, in duct cells and hepatocytes of the liver, and in epithelial cells of the intestine. In all these cell-types epiplakin showed a localization corresponding to keratin filaments of the cytoskeleton. Moreover, it was shown that several domains of epiplakin interact with those keratin variants, namely keratin 8 (K8), K18, and K19, that are most abundant in simple epithelia. Interestingly, epiplakin and K8 showed identical expression levels in all stress models performed. While mice lacking epiplakin react similar to normal mice during an inflammation in the intestine, they clearly show increased liver damages in those diseases which lead to a significantly upregulation of epiplakin and K8 expression. In addition, livers devoid of epiplakin showed an increased number of cells displaying keratin aggregates, indicating impaired disease- induced keratin network reorganization. A similar finding could be made in the stressed pancreas of mice lacking epiplakin, which suffered from a more severe inflammation of their pancreas compared to their epiplakin-expressing littermates. In accordance with these data, cells isolated from epiplakin- free livers showed an increased number of keratin aggregates in combination with cell death when K8 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 reorganizations. We have strong evidence that the enhanced formation of keratin aggregations in the absence of epiplakin is the main 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. Therefore it is conceivable that the absence of epiplakin might hold responsible for the adverse outcome of various pathologies of the gastrointestinal tract.