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.

Public Health Relevance

Several thousand fatal cases of acute liver failure occur annually in the U.S. and acetaminophen toxicity accounts for half of the cases. Our research will elucidate a pathway to mitochondrial toxicity and hepatotoxicity, relevant to multiple causes of liver injury, which has great promise in identifying therapeutic targets to prevent or treat these conditions.

Agency
National Institute of Health (NIH)
Institute
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Type
Research Project (R01)
Project #
5R01DK067215-12
Application #
9232141
Study Section
Hepatobiliary Pathophysiology Study Section (HBPP)
Program Officer
Serrano, Jose
Project Start
2015-04-10
Project End
2020-02-28
Budget Start
2017-03-01
Budget End
2018-02-28
Support Year
12
Fiscal Year
2017
Total Cost
$428,846
Indirect Cost
$168,939
Name
University of Southern California
Department
Internal Medicine/Medicine
Type
Schools of Medicine
DUNS #
072933393
City
Los Angeles
State
CA
Country
United States
Zip Code
90032
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Pyzik, Michal; Rath, Timo; Kuo, Timothy T et al. (2017) Hepatic FcRn regulates albumin homeostasis and susceptibility to liver injury. Proc Natl Acad Sci U S A 114:E2862-E2871
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Huo, Yazhen; Win, Sanda; Than, Tin Aung et al. (2017) Antcin H Protects Against Acute Liver Injury Through Disruption of the Interaction of c-Jun-N-Terminal Kinase with Mitochondria. Antioxid Redox Signal 26:207-220
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Iorga, Andrea; Dara, Lily; Kaplowitz, Neil (2017) Drug-Induced Liver Injury: Cascade of Events Leading to Cell Death, Apoptosis or Necrosis. Int J Mol Sci 18:
Suda, Jo; Dara, Lily; Yang, Luoluo et al. (2016) Knockdown of RIPK1 Markedly Exacerbates Murine Immune-Mediated Liver Injury through Massive Apoptosis of Hepatocytes, Independent of Necroptosis and Inhibition of NF-?B. J Immunol 197:3120-3129
Dara, Lily; Liu, Zhang-Xu; Kaplowitz, Neil (2016) Questions and controversies: the role of necroptosis in liver disease. Cell Death Discov 2:16089
Smith, Andrew K; Petersen, Brenden K; Ropella, Glen E P et al. (2016) Competing Mechanistic Hypotheses of Acetaminophen-Induced Hepatotoxicity Challenged by Virtual Experiments. PLoS Comput Biol 12:e1005253

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