Cellular Mechanisms of Hepatotoxicity.

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DESCRIPTION (provided by applicant): We have identified a novel and extremely important interplay between c-jun-N-terminal kinase (JNK) and mitochondria, outer membrane protein Sab, which leads to mitochondria dysfunction and increased ROS production, which in turn sustains JNK activation in a self-amplifying cycle, leading to hepatocellular necrosis from acetaminophen (APAP) and apoptosis from TNF, ER stress, or saturated fatty acids. Based upon our work accomplished and preliminary results we are poised to advance our understanding of the role of Sab in hepatotoxicity. The specific aims are: 1) Determine the role of phosphorylation of Sab in mediating JNK dependent hepatotoxicity using Sab knockout mice and expression of mutated Sab forms. Hypothesis: JNK mediated phosphorylation of Sab is required for impairment of mitochondrial function and toxicity. We will verify that liver specific conditional knockout of Sab is hepatoprotective in various JNK dependent toxicities in vitro and in vivo. We will then express mutated Sabs (phosphorylation incompetent and phosphomimetic mutants) and determine the impact on mitochondrial function and susceptibility to toxicity. 2) Determine the intramitochondrial signaling pathway downstream of Sab: role of mitochondrial c-Src and Dok-4. Hypothesis: JNK phosphorylation of Sab leads to dephosphorylation of intramitochondrial active c-Src which inactivates the continuously required active c-Src needed to maintain electron transport; this leads to inhibition of oxidative phosphorylation and increased ROS, key steps in JNK-dependent hepatotoxicities. We plan to identify and localize the regulators of the Src family in mitochondria and their association with Sab and Dok-4 (mitochondrial Src kinase docking protein) and the role of Src dysregulation in mitochondrial ROS production. Then we will extend out studies of mitochondrial Src dysregulation from APAP to other models of JNK dependent hepatotoxicity in vitro and in vivo. 3) Determine the mechanism and importance of mitochondrial fission and its interplay with Sab and JNK in mediating hepatotoxicity. Hypothesis: JNK mediated hepatotoxicity depends on the Sab dependent stabilization of MFF, allowing DRP-1 translocation and mitochondrial fission. We have made two important preliminary discoveries. The outer membrane Mitochondrial Fission Factor, MFF, co-immunoprecipitates (IP) with Sab and its expression rapidly increases after APAP. Therefore, we will IP MFF to confirm the association with Sab and determine if P-JNK phosphorylates MFF. We will determine if proteasomal or intramitochondrial degradation accounts for low basal MFF. Then we will knockdown MFF and determine if this affords protection against JNK mediated mitochondrial dysfunction and injury. Similarly, we will assess the effect of knockdown of the other key protein in fission, DRP-1, and confirm the findings by expression of dominant negative DRP-1.