Ethanol exposure promotes the development of alcoholic liver disease (ALD) but the mechanisms underlying ethanol-induced hepatotoxicity remain poorly understood. An important cellular site manifesting damage brought on by ethanol is the mitochondria. Proinflammatory cytokines such as tumor necrosis factor alpha (TNFalpha or TNF) have been linked to many of the associated damage and repair processes seen in ALD. TNFalpha prompts the opening of the mitochondrial permeability transition pore (MPT). The MPT is the regulatable opening of a large, nonspecific pore across the outer and inner mitochondrial membrane. Development of the MPT has been implicated in both necrotic and some forms of apoptotic cell death. Inhibition of the MPT prevents many of the typical manifestations seen during apoptosis. Liver mitochondria isolated from chronically ethanol fed rats are more susceptible to MPT pore opening induced by various triggers than mitochondria isolated from control animals. In preliminary results we demonstrate an enhancement of TNFalpha cytotoxicity in hepatocytes isolated from chronically ethanol-fed rats compared to their pair-fed controls. Similarly, a 48h exposure of HepG2 cells to ethanol enhanced susceptibility to TNFalpha cytotoxicity. The data suggest that ethanol enhances TNFalpha cytotoxicity, at least in part, by promoting the MPT. The general goals of this project are to investigate the mechanisms by which ethanol potentiates TNFalpha induced cytotoxicity. Our working hypothesis is that ethanol inhibits signaling pathways that protect against TNFalpha cytotoxicity and activates pathways that promote the mitochondrial permeability transition. This theory is supported by our preliminary observations that ethanol inhibits P13-kinase dependent BAD phosphorylation induced by TNFalpha and promotes cell death in a p38 MAPK dependent and caspase 8 independent manner. In addition, ethanol exposure sensitizes mitochondria to the MPT and causes an increase in the levels of the peripheral benzodiazepine receptor, a putative component of the MPT pore.
Our SPECIFIC AIMS are to #1) further define the role of ethanol in the alteration of P13-kinase activity and its influence on TNFalpha cytotoxicity and #2) delineate the role and mechanism(s) of activation of p38 MAPK in ethanol plus TNFalpha cytotoxicity; #3) Define the mechanism(s) by which TNF plus ethanol cause the MPT.

Agency
National Institute of Health (NIH)
Institute
National Institute on Alcohol Abuse and Alcoholism (NIAAA)
Type
Research Project (R01)
Project #
1R01AA012897-01A2
Application #
6469999
Study Section
Alcohol and Toxicology Subcommittee 4 (ALTX)
Program Officer
Purohit, Vishnu
Project Start
2002-07-01
Project End
2007-06-30
Budget Start
2002-07-01
Budget End
2003-06-30
Support Year
1
Fiscal Year
2002
Total Cost
$211,017
Indirect Cost
Name
Thomas Jefferson University
Department
Pathology
Type
Schools of Medicine
DUNS #
061197161
City
Philadelphia
State
PA
Country
United States
Zip Code
19107
Shulga, Nataly; Pastorino, John G (2014) Mitoneet mediates TNF?-induced necroptosis promoted by exposure to fructose and ethanol. J Cell Sci 127:896-907
Shulga, Nataly; Pastorino, John G (2014) Hexokinase II binding to mitochondria is necessary for Kupffer cell activation and is potentiated by ethanol exposure. J Biol Chem 289:26213-25
Verma, Manish; Shulga, Nataly; Pastorino, John G (2013) Sirtuin-4 modulates sensitivity to induction of the mitochondrial permeability transition pore. Biochim Biophys Acta 1827:38-49
Neary, Catherine L; Pastorino, John G (2013) Akt inhibition promotes hexokinase 2 redistribution and glucose uptake in cancer cells. J Cell Physiol 228:1943-8
Shulga, Nataly; Pastorino, John G (2012) GRIM-19-mediated translocation of STAT3 to mitochondria is necessary for TNF-induced necroptosis. J Cell Sci 125:2995-3003
Shulga, Nataly; Pastorino, John G (2010) Ethanol sensitizes mitochondria to the permeability transition by inhibiting deacetylation of cyclophilin-D mediated by sirtuin-3. J Cell Sci 123:4117-27
Pastorino, John G; Shulga, Nataly (2008) Tumor necrosis factor-alpha can provoke cleavage and activation of sterol regulatory element-binding protein in ethanol-exposed cells via a caspase-dependent pathway that is cholesterol insensitive. J Biol Chem 283:25638-49
Pastorino, John G; Hoek, Jan B (2008) Regulation of hexokinase binding to VDAC. J Bioenerg Biomembr 40:171-82
Shulga, Nataly; Pastorino, John G (2006) Acyl coenzyme A-binding protein augments bid-induced mitochondrial damage and cell death by activating mu-calpain. J Biol Chem 281:30824-33
Pastorino, John G; Hoek, Jan B; Shulga, Nataly (2005) Activation of glycogen synthase kinase 3beta disrupts the binding of hexokinase II to mitochondria by phosphorylating voltage-dependent anion channel and potentiates chemotherapy-induced cytotoxicity. Cancer Res 65:10545-54

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