Toward understanding the molecular mechanisms that regulate intermediate filament keratin functions in hepatocytes

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Intermediate filaments (IFs) are with microtubules and actin microfilaments the major cytoskeletal components of most mammalian cells. Modifications in K8/18 IFs organization and formation of keratin containing aggregates called Mallory-Denk bodies are common to many human chronic liver diseases such as alcoholic hepatitis, Wilson's disease and liver steatosis in obesity. Recent studies have shown evidences that K8/18 play an important role in the protection of hepatocytes against chronic stress-induced liver injuries. The long-term objective of our project is to determine the molecular mechanism by which K8/18 accomplish their protective role in hepatocytes. More specifically we want to test the hypothesis that K8/18 act as a key regulator in maintaining the delicate balance between pro and anti-apoptotic signals molecules. The short term objective of this proposal is to 1) perform a functional study to directly asses the role of keratins in the anti-apoptotic pathway PI3K/Akt. We will determine how modulation of K8/18 dynamic (expression, phosphorylation on specific sites) affects Akt pathway during the response of cell to Fas and TNF induced apoptosis. 2) There is accumulating evidence that sites and pattern- specific phosphorylation of keratin play a key role in the protection of hepatocytes against stress. Thus, we want to characterize by a proteomic approach the phosphorylation patterns of K8/18 in control hepatocytes and in hepatocytes from mice treated with the hepatotoxic drug griseofulvin. This study will be followed by a functional analysis of the specific K8/18 phosphorylation combination. The characterization of the signaling pathway in which keratins are involved and the study of the modifications that affect keratins (post-translational modification, association with other proteins) during the response of hepatocytes to apoptotic signal will constitute an important step in our understanding of the molecular mechanisms by which keratins accomplish their functions. This will directly lead us to further research on the cross-talk between keratins and cell signaling, and on how keratins affect disease pathogenesis.