Title: Redox Mechanisms of Ischemia Reperfusion Injury in the Liver Hepatic ischemia/reperfusion injury (I/R) is an important clinical problem associated with orthotopic liver transplantation, hepatic resection, and other surgical procedures that transiently alter hepatic blood flow. During the previous two funding cycles, our research has focused on dissecting the mechanisms by which reactive oxygen species (ROS) modulate signaling pathways following reoxygenation injury in the liver and in cell line models of such injury. This proposal focuses on dissecting two ROS-mediated pathways of NF?B activation following liver I/R: reperfusion-initiated c-Src activation and subsequent TNF?-initiated I?B kinase (IKK) activation. We have found that c-Src-mediated activation of NF?B during the acute reperfusion phases of injury is controlled by H2O2-dependent c-Src activation in the endosomal compartment. Our preliminary data on this mechanism suggest that c-Src-mediated NF?B activation establishes a TNF?-dependent pro-inflammatory program that is detrimental to the liver during later phases of I/R injury. We hypothesize that TNF? derived from this c-Src?NF?B pathway in hepatocytes acts to amplify pro-inflammatory TNF? signaling through Kupffer cells. In addition, we have found that TNF? stimulation of NF?B also requires the formation of redox-active endosomes to facilitate TNF receptor (TNFR) complex formation and IKK activation. The existence of two independent redox-regulated mechanisms of NF?B activation, each associated with the endosomal compartment, prompted us to investigate the sources of ROS required for activation of c-Src and TNFR pathways. Our data suggests that NADPH oxidases (Nox) are the primary source of ROS required for activation of these pathways. This proposal will utilize Nox1-, Nox2-, p22phox-, c-Src-, Rac1- and TNF?- deficient mouse models to dissect the redox-dependent components of c-Src- and TNFR-mediated NF?B activation following liver I/R and/or hypoxia/reoxygenation (H/R) in isolated hepatocyte and Kupffer cell culture models. The focus of studies outlined in Aim 1 will be to determine the molecular mechanisms that drive the formation of redox-active endosomes during the reoxygenation phase of injury, to facilitate c-Src activation by Nox.
Aim 2 will investigate the molecular mechanisms by which endosomal Nox controls TNFR1 activation of NF?B, using subcellular fractionation assays and Nox-deficient model systems.
In Aim 3 we will dissect how c- Src activation pathways coordinate downstream TNF? signaling between hepatocytes and Kupffer cells to establish a pro-inflammatory response in the liver. These studies will provide a fundamental understanding of how the subcellular compartmentalization of ROS facilitates NF?B activation in the liver and the manner in which these responses establish a detrimental pro-inflammatory program. Such studies may lead to therapeutic interventions that make it possible to reduce liver injury following I/R by modulating the redox-dependent responses most deleterious to the liver.

Public Health Relevance

Ischemia/reperfusion injury is an important clinical complication associated with liver transplantation, hepatic resection, and other surgical procedures that transiently block blood flow to the liver. This proposal seeks to understand how oxidant-dependent signals influence injury responses in the liver following ischemia/reperfusion and may lead to the development of more effective treatments to reduce this type of injury. The biologic mechanisms studied in this proposal may also be relevant to ischemia/reperfusion injury in other organs and/or other types of ischemic injury (i.e., stroke and cardiac infarct), hence, the proposed research may also enhance of our understanding of ischemic injuries in other organs.

National Institute of Health (NIH)
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Research Project (R01)
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Surgery, Anesthesiology and Trauma Study Section (SAT)
Program Officer
Sherker, Averell H
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University of Iowa
Anatomy/Cell Biology
Schools of Medicine
Iowa City
United States
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Spencer, Netanya Y; Engelhardt, John F (2014) The basic biology of redoxosomes in cytokine-mediated signal transduction and implications for disease-specific therapies. Biochemistry 53:1551-64
Spencer, Netanya Y; Zhou, Weihong; Li, Qiang et al. (2013) Hepatocytes produce TNF-* following hypoxia-reoxygenation and liver ischemia-reperfusion in a NADPH oxidase- and c-Src-dependent manner. Am J Physiol Gastrointest Liver Physiol 305:G84-94
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