Grant: Discovery Grants Program - Individual

Intermediate filaments (IFs) are with microtubules and actin microfilaments the major cytoskeletal components of most mammalian cells. Keratins are the IF proteins that are expressed in epithelial cells. A gene-targeting approach has been necessary to unravel the roles of keratin 8 and 18 in hepatocytes, the epithelial cells of the liver. For instance, hepatocytes from mice carrying K8 or K18 mutations that disrupt IF network structure or affect their phosphorylation pattern, and K8- or K18-null mice are highly susceptible to apoptosis induced by hepatotoxic agents. Thus K8/18 are proposed to play an important role in the protection of epithelial cells against stress-induced by toxicants. The mechanisms by which K8/18 accomplish their protective role is far from being fully understood. The long-term objective of our research program is to unravel the crucial mechanisms by which keratins contribute to the protection of epithelial cells against toxic stress. We hypotheses that they play a direct role in the regulation of major signaling pathways involved in cell survival and cell death. The short term objective of this proposal is 1) by using in vitro models and molecular functional approaches, to determine how K8/18 interacts with Akt isoforms and NF-kB, key members of many survival processes. We hypotheses that keratins play a role in the cellular localization of these key regulatory proteins through direct interaction and possible sequestration. 2) We will determine how the modulation of K8/18 phosphorylation, a post-translational modification known to protect epithelial cells against toxic stress, regulates the activity of the signaling pathways. We speculate that specific modifications in K8/18 phosphorylation will modulate the activity of Akt isoforms. We will also determine if Akt play a role in K8/18 phosphorylation by unraveling the influence of each Akt isoforms on WT K8/K18 phosphorylation and dynamic. The work will be realized on different epithelial cell lines that are chosen for the presence of K8/18 and their level of resistance to apoptosis. The study will involve the use of biochemical and molecular tools as well as confocal microscopy analysis. The characterization of how K8/18 interact with major pro-survival signaling pathway such as Akt and NF-kB will constitute an important step in our understanding of the molecular mechanisms by which keratins accomplish their protective functions. This will directly lead to an understanding of the cross-talk between keratins and cell signaling, and consequently to a better understanding on how keratins affect disease pathogenesis.